The significance of cancer stem cells (CSCs) in initiation and progression of colon cancer (CC) has been established. In this study, we investigated the utility of measuring mRNA expression levels of CSC markers EpCAM, LGR5 and LGR4 for predicting survival outcome in surgically treated CC patients. Expression levels were determined in 5 CC cell lines, 66 primary CC tumors and 382 regional lymph nodes of 121 CC patients. Prognostic relevance was determined using Kaplan-Meier survival and Cox regression analyses. CC patients with lymph nodes expressing high levels of EpCAM, LGR5 or LGR4 (higher than a clinical cutoff of 0.07, 0.06 and 2.558 mRNA copies/18S rRNA unit, respectively) had a decreased mean survival time of 32 months for EpCAM and 42 months for both LGR5 and LGR4 at a 12-year follow-up (p = 0.022, p = 0.005 and p = 0.011, respectively). Additional patients at risk for recurrence were detected when LGR5 was combined with the biomarkers CXCL17 or CEA plus CXCL16. In conclusion, the study underscores LGR5 as a particularly useful prognostic biomarker and illustrates the strength of combining biomarkers detecting different subpopulations of cancer cells and/or cells in the tumor microenvironment for predicting recurrence.

Chemokines are important in the development and progression of tumors. We investigated the expression of CXCL14 and CXCL16 in colon cancer. Expression of mRNA was assessed in primary tumors and lymph nodes and CXCL16 mRNA levels were correlated to patient’s survival. Protein expression was investigated by two-color immunofluorescence and immunomorphometry. CXCL14 and CXCL16 mRNA levels and protein expression were significantly higher in colon cancer primary tumors compared to apparently normal colon tissue. Positive cells were tumor cells, as revealed by anti-CEA and anti-EpCAM staining. CXCL16, but not CXCL14, mRNA levels were significantly higher in hematoxylin and eosin positive (H&E(+)) compared to H&E(−) colon cancer lymph nodes or control nodes (P < 0.0001). CXCL16 mRNA was expressed in 5/5 colon cancer cell lines while CXCL14 was expressed significantly in only one. Kaplan-Meier analysis revealed that colon cancer patients with lymph nodes expressing high or very high levels (7.2 and 11.4 copies/18S rRNA unit, respectively) of CXCL16 mRNA had a decreased mean survival time of 30 and 46 months at the 12-year follow-up (P = 0.04, P = 0.005, respectively). In conclusion, high expression of CXCL16 mRNA in regional lymph nodes of colon cancer patients is a sign of a poor prognosis.

The utility of mRNA and protein determinations of G protein-coupled receptor 35, that is, GPR35a (GPR35 V1) and GPR35b (GPR35 V2/3), as indicators of outcome for colon cancer patients after curative surgery was investigated. Expression levels of V1 and V2/3 GPR35, carcinoembryonic antigen and CXCL17 mRNAs were assessed in primary tumours and regional lymph nodes of 121 colon cancer patients (stage I–IV), colon cancer cell lines and control colon epithelial cells using real-time quantitative reverse transcriptase-polymerase chain reaction. Expression of G protein-coupled receptor 35 was investigated by two-colour immunohistochemistry and immunomorphometry. GPR35 V2/3 mRNA, but not V1 mRNA, was expressed in colon cancer cell lines, primary colon tumours and control colon epithelial cells. Haematoxylin and eosin positive (H&E(+)), but not H&E(–), lymph nodes expressed high levels of GPR35 V2/3 mRNA (P<0.0001). GPR35b and carcinoembryonic antigen proteins were simultaneously expressed in many colon cancer tumour cells. Kaplan–Meier and hazard ratio analysis revealed that patients with lymph nodes expressing high levels of GPR35 V2/3 mRNA and, in particular, in the group of patients with lymph nodes also expressing carcinoembryonic antigen mRNA, had a short disease-free survival time, 67 months versus 122 months at 12-year follow-up (difference: 55 months, P = 0.001; hazard ratio: 3.6, P = 0.002). In conclusion, high level expression of G protein-coupled receptor 35 V2/3 mRNA in regional lymph nodes of colon cancer patients is a sign of poor prognosis.

G protein-coupled receptor 55 (GPR55) probably plays a role in innate immunity and tumor immunosurveillance through its effect on immune cells, such as T cells and NK cells. In this study, the prognostic value of GPR55 in colon cancer (CC) was investigated. mRNA expression levels of GPR55 were determined in 382 regional lymph nodes of 121 CC patients with 12 years observation time after curative surgery. The same clinical material had previously been analyzed for expression levels of CEA, CXCL16, CXCL17, GPR35 V2/3 and LGR5 mRNAs. Clinical cutoffs of 0.1365 copies/18S rRNA unit for GPR55 and 0.1481 for the GPR55/CEA ratio were applied to differentiate between the high- and low-GPR55 expression groups. Kaplan–Meier survival analysis and Cox regression risk analysis were used to determine prognostic value. Improved discrimination between the two groups was achieved by combining GPR55 with CEA, CXCL16 or CXCL17 compared with GPR55 alone. The best result was obtained using the GPR55/CEA ratio, with an increased mean survival time of 14 and 33 months at 5 and 12 years observation time, respectively (p = 0.0003 and p = 0.003) for the high-GPR55/CEA group. The explanation for the observed improvement is most likely that GPR55 is a marker for T cells and B cells in lymph nodes, whereas CEA, CXCL16 and CXCL17, are markers for tumor cells of epithelial origin.

Introduction: Aluminium [Al(III)] is the third most abundant element as well as the most abundant metal in the Earth's crust. The human population is exposed to Al throughout daily life; in drinking water, food, via application of antiperspirants, use of antacids and vaccination. However, aluminum exposure has been linked to a variety of pathological disorders like anemia, osteomalacia , hepatic disorder, and neurological disorder. In addition, studies report that aluminum levels are higher in tumors, including breast cancers than in surrounding normal tissues. This suggests that aluminum accumulation may provide some selective advantage to the tumor. Methods/Results:To determine how aluminum affects cells that reside in mammary tissue, we studied the in vitro effects of Al exposure on human mammary epithelial cells and mesenchymal cells. Cells used in our study included hTERT-immortalized human mammary epithelial cells (HMECs), the immortalized human mammary epithelial cell line (MCF10A), hTERT-immortalized reduction mammary fibroblasts (RMF/EG) and a dermal-derived human fibroblast cell line (MSU1.1). Using the MTT assay we found that AlCl3 and AlOH3 salts inhibit the growth of the mammary epithelial cells causing complete inhibition of proliferation at doses around 1mM and decreased proliferation was observed at doses as low as 12.8 uM. Surprisingly at these doses, Al salts caused dose-dependent increase in proliferation the mesenchymal cells. The proliferative effect of Al on mesenchymal cells was confirmed using clonogenic assay. Flow cytometric evaluation of cell cycle using PI/BrdU incorporation demonstrated that in MSU1.1 cells exposed to AlOH3 for 48 hrs, the percentage of cells in S phase of the cell cycle nearly doubled as compared to untreated cells (P <0.05). Similar results were seen with AlCl3 treatment of MSU1.1 and AlOH3/AlCl3 treatment of RMF/EG cells. Conclusions: These data indicate that while Al salts induce dose dependent cytotoxicity in human epithelial cell lines, they induce proliferation of mesenchymal cells. The tumor microenvironment, including a proliferative stroma, is known to facilitate mammary carcinogenesis. Our data suggest that Al accumulation could promote the development of a permissive microenvironment. We are currently seeking to determine the mechanism for the differential effect of Al in cells and whether this effect influences transformation or carcinogenesis. Citation Format: Manar A. AbdelMageed, Parthena Foltopoulou, Monica Betancur-Boissel, Elizabeth A. McNiel. Differential effects of aluminum exposure on epithelial and mesenchymal cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1157. doi:10.1158/1538-7445.AM2014-1157