Abstract Introduction
The existence of cancer stem cells (CSCs) has been associated with tumor initiation, therapy resistance, tumor relapse, angiogenesis, and metastasis. Curcumin, a plant ployphenol, has several anti-tumor effects and has been shown to target CSCs. Here, we aimed at evaluating (i) the mechanisms underlying the aggravated migration potential of breast CSCs (bCSCs) and (ii) the effects of curcumin in modulating the same.
The migratory behavior of MCF-7 bCSCs was assessed by using cell adhesion, spreading, transwell migration, and three-dimensional invasion assays. Stem cell characteristics were studied by using flow cytometry. The effects of curcumin on bCSCs were deciphered by cell viability assay, Western blotting, confocal microscopy, and small interfering RNA (siRNA)-mediated gene silencing. Evaluations of samples of patients with breast cancer were performed by using immunohistochemistry and flow cytometry.
Here, we report that bCSCs are endowed with aggravated migration property due to the inherent suppression of the tumor suppressor, E-cadherin, which is restored by curcumin. A search for the underlying mechanism revealed that, in bCSCs, higher nuclear translocation of beta-catenin (i) decreases E-cadherin/beta-catenin complex formation and membrane retention of beta-catenin, (ii) upregulates the expression of its epithelial-mesenchymal transition (EMT)-promoting target genes (including Slug), and thereby (iii) downregulates E-cadherin transcription to subsequently promote EMT and migration of these bCSCs. In contrast, curcumin inhibits beta-catenin nuclear translocation, thus impeding trans-activation of Slug. As a consequence, E-cadherin expression is restored, thereby increasing E-cadherin/beta-catenin complex formation and cytosolic retention of more beta-catenin to finally suppress EMT and migration of bCSCs.
Cumulatively, our findings disclose that curcumin inhibits bCSC migration by amplifying E-cadherin/beta-catenin negative feedback loop.
Breast cancer is the most common form of cancer diagnosed in women. In 2013, breast cancer accounted for 29% of all new cancer cases and 14% of all cancer deaths among women worldwide [1]. Breast cancer-related mortality is associated with the development of metastatic potential of the primary tumor [2]. Given this high rate of incidence and mortality, it is critical to understand the mechanisms behind metastasis and identify new targets for therapy. For the last few decades, various modalities of cancer therapy were being investigated. But the disease has remained unconquered, largely because of its invasive nature.
Amidst the research efforts to better understand cancer progression, there has been increasing evidence that hints at a role for a subpopulation of tumorigenic cancer cells, termed cancer stem cells (CSCs), in metastasis formation [3]. CSCs are characterized by their preferential ability to initiate and propagate tumor growth and their selective capacity for self-renewal and differentiation into less tumorigenic cancer cells [4]. There are reports which demonstrate that CSCs are enriched among circulating tumor cells in the peripheral blood of patients with breast cancer [5]. Moreover, recent studies show that epithelial-mesenchymal transition (EMT), an early step of tumor cell migration, can induce differentiated cancer cells into a CSC-like state [6]. These observations have established a functional link between CSCs and EMT and suggest that CSCs may underlie local and distant metastases by acquiring mesenchymal features which would greatly facilitate systemic dissemination from the primary tumor mass [7]. Taken together, these studies suggest that CSCs may be a critical factor in the metastatic cascade. Now, the incurability of the malignancy of the disease raises the question of whether conventional anti-cancer therapies target the correct cells since the actual culprits appear to be evasive of current treatment modalities.
Studies focusing on the early steps in the metastatic cascade, such as EMT and altered cell adhesion and motility, have demonstrated that aggressive cancer progression is correlated with the loss of epithelial characteristics and the gain of migratory and mesenchymal phenotype [8], for which downregulation of E-cadherin is a fundamental event [9]. A transcriptional consequence of the presence of E-cadherin in epithelial cells can be inferred from the normal association of E-cadherin with -catenin in adherens junctions. This association prevents -catenin transfer to the nucleus and impedes its role as a transcriptional activator, which occurs through its interaction mainly with the TCF (T-cell factor)-LEF (lymphoid enhancer factor) family of transcription factors but also with other DNA-binding proteins [10]. Accordingly, the involvement of -catenin signaling in EMTs during tumor invasion has been established [11]. Aberrant expression of -catenin has been reported to induce malignant pathways in normal cells [12]. In fact, -catenin acts as an oncogene and modulates transcription of genes to drive cancer initiation, progression, survival, and relapse [12]. All of the existing information regarding abnormal expression and function of -catenin in cancer makes it a putative drug target [12] since its targeting will negatively affect both tumor metastasis and stem cell maintenance. Transcriptional target genes of -catenin involve several EMT-promoting genes, including Slug. Expression of Slug has been shown to be associated with breast tumor recurrence and metastasis [13-15]. Pro-migratory transcription factor Slug (EMT-TF), which can repress E-cadherin, triggers the steps of desmosomal disruption, cell spreading, and partial separation at cell-cell borders, which comprise the first and necessary phase of the EMT process [16].
Recently, the use of natural phytochemicals to impede tumor metastasis via multiple targets that regulate the migration potential of tumor cells has gained immense importance [17]. In this regard, curcumin, a dietary polyphenol, has been studied extensively as a chemopreventive agent in a variety of cancers, including those of the breast, liver, prostate, hematological, gastrointestinal, and colorectal cancers, and as an inhibitor of metastasis [18]. In a recent report, curcumin was shown to selectively inhibit the growth and self-renewal of breast CSCs (bCSCs) [19]. However, there are no reports regarding the contribution of curcumin in bCSC migration.
The present study describes (i) the mechanisms governing the augmented migration potential of bCSCs, which (ii) possibly associates with tumor aggressiveness and is largely attributable to the inherent downregulation of the anti-migratory tumor suppressor protein, E-cadherin, in bCSCs, and (iii) the role of curcumin in modulating the same. A search for the upstream mechanism revealed higher nuclear translocation and transcriptional activity of -catenin resulting from disruption of E-cadherin/-catenin complex formation in bCSCs in comparison with non-stem tumor cells. Upregulation of nuclear -catenin resulted in the augmentation of Slug gene expression that, in turn, repressed E-cadherin expression. In contrast, exposure to curcumin inhibited the nuclear translocation of -catenin, thereby hampering the activation of its EMT-promoting target genes, including Slug. Resultant upregulation of E-cadherin led to increase in E-cadherin/-catenin complex formation, which further inhibited nuclear translocation of -catenin. As a consequence, the E-cadherin/-catenin negative feedback loop was amplified upon curcumin exposure, which reportedly inhibits EMT on one hand and promotes cell-cell adherens junction formation on the other. These results suggest that curcumin-mediated inhibition of bCSC migration may be a possible way for achieving CSC-targeted therapy to better fight invasive breast cancers.
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