Colorectal cancer cell-derived microRNA200 modulates the resistance of adjacent blood endothelial barriers in vitro.

Since cancer cells, when grown as spheroids, display drug sensitivity and radiation resistance patterns such as seen in vivo we recently established a three­dimensional (3D) in vitro model recapitulating colorectal cancer (CRC)-triggered lymphatic endothelial cell (LEC)­barrier breaching to study mechanisms of intra­/extravasation. CRC metastasizes not only through lymphatics but also through blood vessels and here we extend the 3D model to the interaction of blood endothelial cells (BECs) with naïve and 5­fluorouracil (5­FU)­resistant CRC CCL227 cells. The 3D model enabled quantifying effects of tumour­derived microRNA200 (miR200) miR200a, miR200b, miR200c, miR141 and miR429 regarding the induction of so-called 'circular chemorepellent­induced defects' (CCIDs) within the BEC­barrier, which resemble gates for tumour transmigration. For this, miR200 precursors were individually transfected and furthermore, the modulation of ZEB family expression was analysed by western blotting. miR200c, miR141 and miR429, which are contained in exosomes from naïve CCL227 cells, downregulated the expression of ZEB2, SNAI and TWIST in BECs. The exosomes of 5­FU­resistant CCL227­RH cells, which are devoid of miR200, accelerated CCID formation in BEC monolayers as compared to exosomes from naïve CCL227 cells. This confirmed the reported role of ZEB2 and SNAI in CRC metastasis and highlighted the active contribution of the stroma in the metastatic process. CCL227 spheroids affected the integrity of BEC and LEC barriers alike, which was in agreement with the observation that CRC metastasizes via blood stream (into the liver) as well as via lymphatics (into lymph nodes and lungs). This further validated the CRC/LEC and CRC/BEC in vitro model to study mechanisms of CRC spreading through vascular systems. Treatment of CCL227­RH cells with the HDAC inhibitors mocetinostat and sulforaphane reduced CCID formation to the level triggered by naïve CCL227 spheroids, however, without significantly influencing miR200 expression in CCL227-RH cells.

NFκB-Associated Pathways in Progression of Chemoresistance to 5-Fluorouracil in an In Vitro Model of Colonic Carcinoma.

BACKGROUND: Drug resistance to 5-fluorouracil (5-FU) is a major obstacle in colonic cancer treatment. Activation of nuclear factor-kappa B (NFκB), mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and protein kinase B (AKT) is thought to protect cancer cells against therapy-induced cytotoxicity. MATERIALS AND METHODS: Using cytotoxicity assays and immunoblotting, the impact of inhibitory strategies addressing NFκB, AKT and MAP3K8 in chemoresistance was evaluated in a colonic cancer model in vitro. This model consisted of the cell lines SW480 and SW620, and three subclones with increasing degrees of chemoresistance in order to mimic the development of secondary resistance. RESULTS: NFκB protein p65 was selectively activated in all resistant cell lines. Consequently, several inhibitors of NFκB, MAP3K8 and AKT effectively circumvented this chemoresistance. As a cellular reaction, NFκB inhibition may trigger a feedback loop resulting in activation of extracellular signal-regulated kinase. The results suggest that chemoresistance to 5-FU in this colonic carcinoma model (cell lines SW480 and SW620) is strongly dependent on NFκB activation. The efficacy of MAP3K8 inhibition in our model potentially uncovers a new mechanism to circumvent 5-FU resistance.

Loss of miR-200 family in 5-fluorouracil resistant colon cancer drives lymphendothelial invasiveness in vitro.

Invasive colorectal cancer is associated with poor prognosis requiring treatment with systemic chemotherapies usually including 5-fluorouracil. A consequence of prolonged treatment is the acquisition of resistance eventually resulting in the recurrence of highly metastatic cancer cells. To address the relationship between drug resistance and increased lymphatic metastatic potential, we used a 3D co-culture model of colon tumour cell spheroids of parent CCL227 cells and subclones with gradually increasing resistance against 5-fluorouracil. From each investigated cell line, homogeneous tumour spheroids were generated in the presence of methylcellulose yielding emboli of ∼700 µm diameter. When invasive, tumour spheroids disrupt the continuous lymphendothelial cell (LEC) layer and generate a 'circular chemorepellent-induced defect' (CCID), reminiscent of the entry gates through which tumour emboli intravasate lymphatic vasculature. Here we provide evidence that increasingly chemoresistant colon cancer spheroids were strongly associated with enhanced intravasative properties. In naïve CCL227 spheroids, miR-200 family members were released into exosomes thereby repressing the epithelial to mesenchymal transition-regulating transcription factors ZEB1 and SLUG in LEC. As a consequence of attenuated plasticity and migration of LEC, CCID formation was impaired. Loss of exosomal transferred miR-200c in resistant colon cells rendered LEC more susceptible to pro-migratory signals that were generated and directly transmitted by colon cancer spheroids. This observation indicates a common molecular axis in colon cancer and LEC where miR-200 family members act as regulators of ZEB proteins. The data support the notion that horizontal miR-200 signalling prevents the permeation of cells into adjacent epithelia and contributes to organ integrity.