Elsevier

Cellular Signalling

Volume 26, Issue 3, March 2014, Pages 492-501
Cellular Signalling

β-Catenin, a Sox2 binding partner, regulates the DNA binding and transcriptional activity of Sox2 in breast cancer cells

https://doi.org/10.1016/j.cellsig.2013.11.023Get rights and content

Highlights

  • Sox2 binds to β-catenin in MCF7 cells.

  • The functional relationship between Sox2 and β-catenin has been studied.

  • β-Catenin suppresses the transcriptional activity of Sox2 in a small subset of BC cells.

  • The potential clinical implications of our findings have been discussed.

Abstract

Sox2, an embryonic stem cell marker, has been recently implicated in the pathogenesis of breast cancer (BC). Using liquid chromatography–mass spectrometry and co-immunoprecipitation, we identified β-catenin as a Sox2 binding partner in MCF7 cells. The interaction between Sox2 and β-catenin was substantially different between the two cell subsets separated based on their differential responsiveness to a Sox2 reporter. Specifically, while β-catenin binds to Sox2 in the nuclear fraction of cells showing reporter-responsiveness (i.e. RR cells), this interaction was not detectable in those that were reporter-unresponsive (i.e. RU cells). In RR but not in RU cells, siRNA knockdown of β-catenin significantly upregulated the Sox2 transcriptional activity, enhanced its DNA binding and increased the expression of its target genes. Correlating with these findings, while inhibition of β-catenin significantly downregulated the mammosphere formation efficiency in RU cells, this treatment paradoxically increased that of RR cells. To conclude, we identified that β-catenin is an important binding partner of Sox2 and a regulator of its transcriptional activity in a small subset of BC cells. The interaction between Sox2 and β-catenin provides a novel mechanism underlying the functional dichotomy of BC cells, which carries potential therapeutic implications.

Introduction

Sex determining region Y-Box 2 (Sox2) is a transcriptional factor known to play key roles in maintaining the pluripotency of embryonic stem cells (ESCs) [1], [2], [3]. The contribution of Sox2 to stemness is underscored by the fact that it is one of the four genes involved in the conversion of fibroblasts into inducible pluripotent stem cells [4], [5], [6]. Normally, the expression of Sox2 is restricted in ESCs and somatic stem cells [2], [7], [8]. In recent years, Sox2 has been found to be aberrantly expressed in several types of solid tumors including breast cancer (BC) [9], [10], [11], [12], [13], [14], [15], [16], [17]. Accumulating evidence has supported the concept that Sox2 is biologically and clinically important in these cancer types. For instance, Sox2 expression is known to correlate with increased migration, proliferation, and tumorigenicity in lung carcinomas [9], [18], [19], [20] and glioblastomas [11]. In BC, Sox2 expression was found in tumors representative of all 4 major molecular subtypes, including luminal A, luminal B, Her2-positive and basal-like [21]. In one study, a significant correlation between the level of Sox2 protein expression in BC detectable by immunohistochemistry and a worse clinical outcome was found [22]. Nevertheless, only a relatively small number of functional studies have provided direct evidence that Sox2 is important in the biology of BC. To this end, we have identified 3 studies in which the functional roles of Sox2 in BC were directly demonstrated. In one study, enforced expression of Sox2 in MCF7 cells was found to significantly increase their efficiency of mammosphere formation [21]. In two other studies, it was found that siRNA knockdown of Sox2 significantly reduces the proliferation of MCF7 cells [11], [23].

To further understand the functional significance of Sox2 in BC, we previously assessed if Sox2 is transcriptionally active in BC cell lines. To achieve this goal, we transfected a Sox2 reporter construct into MCF7 and ZR751, two estrogen receptor-positive BC cell lines, as detailed in one of our recent publications [24]. Using the expression of luciferase and/or green fluorescence protein (GFP) as the read-outs, we were able to demonstrate the existence of two distinct cell subsets in both cell lines. Specifically, while most cells did not show responsiveness to the Sox2 reporter (labeled RU cells), approximately 10% of the cells from these two cell lines expressed luciferase and GFP (labeled RR cells). In support that the existence of these two cell subsets is biologically significant, we found that RR and RU cells are significantly different with respect to their efficiencies in forming colonies in soft agar and mammospheres [24]. Our findings strongly suggest that the transcriptional status of Sox2, and thus, the mechanism involved in regulating this biochemical property, is highly important and relevant to the biology of BC. To our knowledge, how the transcriptional activity of Sox2 is regulated in cancer cells has not been comprehensively examined. Nonetheless, studies of Sox2 in ESCs have provided some clues to this question. Specifically, PARP1, one of the Sox2 binding proteins, has been shown to regulate the transcriptional activity of Sox2 in mouse ESCs [25]. Thus, it is possible that Sox2 binding proteins in cancer cells also can regulate the transcriptional activity of Sox2 in these cells.

To test the hypothesis that Sox2 binding proteins can serve as regulators of its transcriptional activity in BC cells, we performed liquid chromatography coupled with tandem mass spectrometry (LC–MS) to comprehensively profile Sox2 binding partners in MCF7 cells. We found that β-catenin is one of the Sox2 binding proteins. Importantly, β-catenin was found to inhibit the transcriptional activity of Sox2, but this regulation was present in RR but not in RU cells. Our findings have shed light into a novel mechanism underlying the functional dichotomy of BC cells. The potential clinical implications of our findings will be discussed.

Section snippets

Cell culture

MCF7, a human BC cell line, was purchased from American Type Culture Collection (ATCC, Rockville, MD) and cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS) (Sigma, Oakville, ON, Canada) under an atmosphere of 5% CO2 at 37 °C.

Generation of stable cell lines transfected with the Sox2 reporter

MCF7 cells stably expressing the Sox2 reporter were generated as described previously [24]. The Sox2 reporter contained three tandem-repeats of Sox2 regulatory region 2

β-Catenin is a Sox2 binding protein

We hypothesized that the transcriptional activity of Sox2 in BC cells is regulated by its binding partners. To this end, we performed LC–MS to profile these proteins in MCF7 cells. To optimize the yield of our assay, we transfected MCF7 cells with a FLAG-tagged Sox2 vector, such that we were able to capture a relatively large quantity of Sox2 protein and its binding partners using anti-FLAG M2 affinity gel. In view of the fact that the RR and RU cell subsets have drastically different

Discussion

While the biological importance of Sox2 in stem cell biology has been well established, its significance in cancer is incompletely understood. In one of our recent publications [24], we examined the status of Sox2 transcriptional activity in breast cancer cells. Intriguingly, in two estrogen receptor-positive BC cell lines, MCF7 and ZR751, we found that the Sox2 transcriptional activity was heterogeneous, with a small cell subset being responsive to the Sox2 reporter (i.e. RR cells) and the

Conflict of interest

No potential conflicts of interestwere disclosed.

Financial support

This study was funded by the Canadian Institutes of Health Research and the Alberta Cancer Foundation awarded to R L. FW was awarded the Alberta Cancer Foundation Cancer Research Postdoctoral Fellowship. KJ is a recipient of the CIHR Vanier Canada Graduate Scholarship.

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