TRAIL receptor upregulation and the implication of KRAS/BRAF mutations in human colon cancer tumors Eftychia Oikonomou 1 , Vivian Kosmidou 1 , Anastasia Katseli 2 , Konstantinos Kothonidis 2 , Despina Mourtzoukou 3 , George Kontogeorgos 3 , Ladislav Andera 4 , Georgios Zografos 2 and Alexander Pintzas 1 * 1 Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Athens, Greece 2 Third Department of Surgery, General Hospital of Athens G. Gennimatas, Athens, Greece 3 Department of Pathology, General Hospital of Athens G. Gennimatas, Athens, Greece 4 Laboratory of Cell Signaling and Apoptosis, Institute of Molecular Genetics, v.v.i., Czech Academy of Sciences, 1083 Videnska, CZ-14220 Prague 4, Czech Republic TRAIL raises hopes as a promising anti-tumor agent due to its se- lectivity toward cancer cells. Higher expression of its pro-death receptors TRAIL-R1 (DR4) and TRAIL-R2 (DR5) attenuates higher sensitivity to TRAIL-induced apoptosis, and represents a marker for better cancer prognosis and treatment. Since receptor availability can be analogous to ligand efficacy, we performed RT- PCR analysis of DR4 and DR5 in 51 colon cancer biopsy speci- mens and respective normal mucosa, while 11 of these tumors were determined immunohistochemically for protein expression. Transcriptional analysis showed that DR4 and DR5 were signifi- cantly upregulated in 37 and 47% of the tumor samples respec- tively, while both DR4 and DR5 were coinstantaneously upregu- lated in 31% of the samples analyzed. Positive transcriptional reg- ulation of DRs was recorded as early as Dukes’ A stage. Furthermore, protein expression analysis yielded results compara- ble to DR4 and DR5 increased mRNA levels. Possible contributing events to DR upregulation involve presence of frequent oncogenic mutations in the MAPK pathway, and was investigated by direct sequencing in all 51 tumors. Samples (6/8) hosting either a KRAS G12V or BRAF V600E mutation, significantly amplified the upregulated expression of DR4 and DR5, showing strong inter- relation between overexpression and presence of oncogenic KRAS/ BRAF mutations. In the light of recent data concerning TRAIL receptor distribution, we contribute further by presenting DR5 as the most frequently upregulated DR in colon cancer. Fur- thermore, oncogenic mutations may directly or indirectly enhance DR expression, potentially sensitizing these tumors to TRAIL- based therapies. ' 2009 UICC Key words: BRAF V600E ; colon cancer; DR5; KRAS G12/13 ; TRAIL Apoptosis has profound implications in developmental biology and tissue homeostasis, since cell number can be regulated by fac- tors that influence cell survival, or control proliferation and differ- entiation. Deregulation of such mechanisms can lead to growth advantage in cancer cells. Most sporadic colorectal cancers (CRCs) are thought to develop from benign adenomas. Progres- sion from normal epithelium through adenoma to colorectal carci- noma is characterized by accumulated abnormalities to particular genes that will ultimately coerce the tumor to invade into sur- rounding tissue and metastasize. 1 In patients with established lymph node metastasis (Dukes’ C), adjuvant fluorouracil-based chemotherapy after surgical resection improves survival. Failure in normal apoptotic pathways (pro- grammed cell death) during carcinogenesis limits the efficacy of anti-cancer drugs or radiotherapy. Consequently, intrinsic as well as acquired resistance to chemotherapeutic drugs remains a major problem. Prominent among cell surface molecules capable of ini- tiating and tightly controlling apoptosis in cancer cells is the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Tumour necrosis factor (TNF) family members TRAIL and FasL induce apoptosis by binding to their corresponding cell-surface receptors DR4, DR5 and FAS, respectively. TRAIL is expressed at the mRNA level in most tissues of the human body. Although TRAIL is mostly a membrane-acting protein, small quantities of its soluble form can also be detected. 2 The focus on TRAIL as a potential therapeutic agent became obvious due to its differential sensitivity observed between normal and cancerous cells, 3,4 whereas its major advantage lies within its ability to trigger tumor cell apoptosis in a variety of cancers independent of p53 status. 5,6 In preclinical models, it has no adverse effects on normal tissue and shows anti-tumor activity. 3,7–9 In addition, TRAIL acts syn- ergistically with chemotherapeutic drugs and can overcome drug resistance in tumor cell lines and animal models. 10–12 Immunohistochemical studies have demonstrated that the pro- apoptotic DR4 and DR5 are expressed in normal colon mucosa as well as colorectal adenomas and carcinomas, and their expression was increased in malignant versus normal cells. In comparison, there is a marked increase in sensitivity to TRAIL-induced apo- ptosis associated with progression from benign to malignant tumor with the assumption that sensitivity to TRAIL could be acquired early in colorectal tumorigenesis during the formation of ade- noma. 13–16 Nonetheless, in some cases, DR receptor expression does not correlate to TRAIL sensitivity in vitro since sensitivity is also controlled by intracellular regulators of apoptosis and should be taken into account when investigating the contribution of DR expression with regard to TRAIL sensitivity. 17 FAS receptor is highly expressed in normal human colonic epithelial cell and its expression is progressively decreased during tumor progression from normal epithelium to adenocarcinoma in about 50% of the cases. 18,19 As a result, sensitivity of colon cancer cells to FasL is significantly reduced. Receptor availability is an important requirement for sensitizing colon cancer cells to TRAIL-mediated apoptosis. DR4 and DR5 expression in colon cancer could become a prognostic factor to- ward the development of new patient-tailored treatment strategies. With respect to induction of DR4 and DR5 expression, we have previously shown that oncogenic RAS sensitizes human colon cells to TRAIL-induced apoptosis by upregulating DR4 and DR5 in a MEK-dependent manner. 20 Studies so far have focused on the im- munohistochemical expression and localization of DR4 and DR5 in colon cancer, 13,15,16,21 rather than the degree of receptor upre- gulation using a semiquantitative method such as RT-PCR. Fur- thermore, studies where normal tissue was used for comparison reasons did not directly correspond to the malignant tissue of the patient. 13–16 In contrast, the present study presents a direct com- parison between malignant tissue and the respective normal mu- cosa from each patient, performed by RT-PCR in 51 samples for Grant sponsor: EU; Grant number: LSHC-CT-2006-037278; Grant sponsor: Center Molecular Cellular Immunology; Grant number: 1M6837805001. *Correspondence to: Laboratory of Signal Mediated Gene Expression, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, Vasileos Konstantinou Ave. 48, 11635, Athens, Greece. Fax: 130-210-7273755. E-mail: apint@eie.gr Received 4 November 2008; Accepted after revision 19 May 2009 DOI 10.1002/ijc.24613 Published online 2 June 2009 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 125, 2127–2135 (2009) ' 2009 UICC Publication of the International Union Against Cancer