Abstract
TNF-alpha-related-apoptosis-inducing-ligand (TRAIL) has been explored as a therapeutic drug to kill cancer cells. Cancer cells in the circulation are subjected to apoptosis-inducing factors. Despite the presence of these factors, cells are able to extravasate and metastasize. The homotypic and heterotypic cell-cell interactions in a tumor are known to play a crucial role in bestowing important characteristics to cancer cells that leave the primary site. Spheroid cell culture has been extensively used to mimic these physiologically relevant interactions. In this work, we show that the breast cancer cell lines BT20 and MCF7, cultured as 3D tumor spheroids, are more resistant to TRAIL-mediated apoptosis by downregulating the expression of death receptors (DR4 and DR5) that initiate TRAIL-mediated apoptosis. For comparison, we also investigated the effect of TRAIL on cells cultured as a 2D monolayer. Our results indicate that tumor spheroids are enriched for CD44hiCD24loALDH1hi cells, a phenotype that is predominantly known to be a marker for breast cancer stem cells. Furthermore, we attribute the TRAIL-resistance and cancer stem cell phenotype observed in tumor spheroids to the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway. We show that inhibition of the COX-2/PGE2 pathway by treating tumor spheroids with NS-398, a selective COX-2 inhibitor, reverses the TRAIL-resistance and decreases the incidence of a CD44hiCD24lo population. Additionally, we show that siRNA mediated knockdown of COX-2 expression in MCF7 cells render them sensitive to TRAIL by increasing the expression of DR4 and DR5. Collectively, our results show the effect of the third-dimension on the response of breast cancer cells to TRAIL and suggest a therapeutic target to overcome TRAIL-resistance.
Highlights
In the hematogenous metastatic cascade, cells from the primary tumor enter the peripheral circulation after which they can mimic the leukocyte adhesion cascade to extravasate through the blood vessel wall and establish in a secondary site [1]
Confocal micrographs indicated a strong staining for surface death receptor expression in BT20 (DR4 and DR5) and MCF7 cells (DR4 only) cultured as a monolayer and a weak or no staining in BT20 and MCF7 cells cultured as 3D tumor spheroids (Fig. 1A)
BT20 and MCF7 cells cultured as tumor spheroids were more resistant to TRAIL-mediated apoptosis when compared to BT20 and MCF7 cells cultured as a monolayer
Summary
In the hematogenous metastatic cascade, cells from the primary tumor enter the peripheral circulation after which they can mimic the leukocyte adhesion cascade to extravasate through the blood vessel wall and establish in a secondary site [1]. TRAIL is one such molecule that plays a key role in body’s natural defense mechanism, which is currently being studied in the field of cancer therapy [4,5,6]. The surface expression of death receptors plays a key role in transmitting the apoptosisinducing signal. Several cancer cell lines have been shown to be resistant to TRAIL-mediated apoptosis by decreasing the expression of death receptors [8], internalizing death receptors by constitutive endocytosis [9], upregulating anti-apoptotic proteins such as Bcl-2 [10], activating cellular survival pathways such as PI3K/Akt signaling pathway [11], upregulating decoy receptors [12,13], or downregulating pro-apoptotic proteins such as Caspase 8 [14]. Studying the underlying mechanism behind TRAILresistance exhibited by certain cancer cells could lead to more effective use of TRAIL in anti-cancer therapy
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