Involvement of the WNT/β-catenin pathway in CD44-targeted therapy of colorectal cancer

B32 Background and Aim: Hyaluronic acid (HA) is a component of the extracellular matrix playing a crucial role in cell adhesion, growth, and migration and CD44, the receptor for HA-mediated motility, is over-expressed in a wide variety of cancers. Therefore, CD44-targeted therapy, using HA as a carrier covalently conjugated to drugs, may be a promising method in cancer therapy. We have previously reported the in vitro and in vivo antitumor effect of ONCOFID™-S, a bioconjugate of hyaluronic acid with SN38 (the active metabolite of irinotecan), on preclinical models of coloncarcinoma. Given the role of deregulation or constitutive activation of the Wnt/β-catenin pathway in colorectal cancer initiation and progression, in this study we assessed if the expression of some crucial molecules involved in the signal transmission initiated by Wnt ligands could be modified by ONCOFID™-S.Methods: DHD/TRB cell line, originally established from a 1,2-dimethylhydrazine(DMH)-induced colon adenocarcinoma in syngeneic BDIX rats, was used in the in vitro study. The intracellular distributions of E-cadherin, β-catenin, APC and GSK-3β were analyzed by CLSM. E-cadherin, β-catenin levels were also evaluated by Western blot in soluble or membrane protein extracts. In vivo study was performed inducing tumor by subcutaneous injection of DHD/TRB in syngeneic BDIX immunocompetent rats. Expression and tissue distribution of the Wnt/β-catenin signaling pathway molecules were analyzed ex vivo on explanted tumors by immunohistochemistry. Results: We confirmed that ONCOFID™-S is able to inhibit DHD/TRB cells proliferation in a dose dependent manner. The antiproliferative effect is preceded by a redistribution of Wnt signaling molecules, with translocation of the onco-suppressor APC to the nucleus after 6-24h and decrease of β-catenin nuclear and cytoplasmic accumulation between 24h and 48h. Increased co-localization of the β-catenin/APC and β-catenin/GSK-3s deriving immunofluorescent signals after treatment indicated that the decreased expression of cytosolic β-catenin might be due to an increase of its degradation. Augmented expression of E-cadherin and β-catenin at the cell-cell junctions further supports that the antiproliferative effect recorded might be due to a specific inhibition of the Wnt/β-catenin signaling pathway. In vivo the perilesional treatment with three cycles of ONCOFID™-S inhibited tumor growth more efficiently than the systemic administration of the unconjugated drug, also resulting less toxic. Immunohistochemical analysis of the expression and tissue distribution of E-cadherin, β-catenin, APC and GSK-3β on ex vivo samples indicated that the Wnt/β-catenin pathway reversion might also occur after ONCOFID™-S in vivo treatment.Conclusions: The bioconjugate ONCOFID™-S exerts an antitumor effect on colorectal cancer both in vitro and in vivo inducing modifications of some molecules of Wnt/β-catenin signaling pathway. Thus, we confirm the efficiency of the drug delivery strategy using hyaluronic acid as a carrier targeting its CD44 receptor, granting the development of this approach for treatment of primary and metastatic colorectal cancer.