Background
Tumor invasiveness is a complex process in which malignant cells dissociate and migrate from the primary site of growth, which may eventually lead to the formation of distant metastases [
1]. In many types of solid tumor, it has been shown that epithelial-mesenchymal transition (EMT) is a crucial step for tumor invasiveness [
2,
3]. During EMT, malignant epithelial cells shed their differentiated characteristics (e.g. cell-cell adhesion, apical-basal polarity and immobility) and acquire mesenchymal features (e.g. increased motility and invasiveness) [
4]. The induction of EMT can be triggered by cytokines, such as TGF-β and interleukin (IL)-8, as well as several transcriptional factors including Twist1, Snail, and ZEB [
5‐
9]. Twist1 has been described to be one of the key promoters of EMT and invasiveness in a number of cancer types [
10‐
12]. In several studies, Twist1 was found to be up-regulated by a number of proteins including STAT3 [
13], BMP2 [
14], SRC-1 [
15], MSX2 [
16], NF-κB [
17], and ILK [
18] and down-regulated by miR-580 and CPEB1/2 [
19]. In breast cancer (BC), Twist1 has been found to promote EMT and invasiveness [
5]. A number of immunohistochemical studies have described a significant positive correlation between Twist1 and the metastatic/invasive property of BC [
5‐
8]. In an animal model, siRNA knockdown of Twist1 was found to inhibit BC cells to metastasize to the lungs [
5]. Furthermore, the mechanisms by which Twist1 promotes tumor invasiveness in BC have been extensively examined; down-regulation of E-cadherin [
9] and up-regulation of SET8 [
20], AKT2 [
8], miRNA-10b [
21], IL8 [
22] and PDGFα [
23] have been implicated.
Sox2 (sex determining region Y-box protein 2) is a transcription factor that plays a key role in maintaining the pluripotency of embryonic stem cells [
24‐
26]. The importance of Sox2 in stem cell biology is highlighted by the fact that Sox2 represents one of the 4 genes implicated in the conversion of fibroblasts into inducible pluripotent stem cells [
27,
28]. Recent studies have shown that Sox2 is aberrantly expressed in several types of solid tumors, including BC, lung cancer, prostate cancer, glioblastomas and melanomas [
29‐
33]. The expression of Sox2 detectable by immunohistochemistry has been found to positively correlate with the invasiveness and metastatic potential of several types of solid tumors [
34‐
37]. Nevertheless,
in-vitro studies that directly assess the role of Sox2 in regulating tumor invasiveness are relatively scarce [
35‐
38]. In several types of cancer cells (e.g., gliomas, melanomas and colorectal cancer), knockdown of Sox2 using siRNA was found to decrease invasiveness [
35‐
37]. In one study, enforced expression of Sox2 in MCF7, an estrogen receptor-positive (ER+) BC cell line, was found to increase invasiveness in an
in-vitro assay by approximately 60% [
38]. The mechanisms by which Sox2 regulates the invasiveness of BC cells are largely unknown. For instance, whether the regulatory effects of Sox2 on the invasiveness of BC are linked to regulators of EMT (such as Twist1) has not been examined previously.
In this study, we aimed to further define the roles of Sox2 in regulating the invasiveness of BC cells. In contradiction with the conclusion of a recently published paper [
38], we found that Sox2 suppresses, rather than increases, the invasiveness of MCF7 cells. Furthermore, this biological effect is dependent on the regulation of Twist1 expression by Sox2. When we assessed the roles of Sox2 in the two distinct cell subsets of MCF7 separated based on their differential responsiveness to the
Sox2 reporter, as shown previously [
39], we found that the Sox2-mediated effects on invasiveness in BC is restricted to ‘reporter un-responsive’ (RU) cells. We believe that our results have shed important insights into the biological significance of Sox2 in BC, the invasiveness property of BC, as well as a new level of biological complexity of this type of cancer.
Methods
Cell culture
MCF7 and ZR751 were purchased from American Type Culture Collection (ATCC, Rockville, MD). Both ZR751 and MCF7 cells were maintained 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) and were cultured under an atmosphere of 5% CO2 at 37°C.
Generation of stable cell lines
Stable cells expressing the
Sox2 GFP reporter were generated as previously described [
39]. Cells stably expressing the
Sox2 GFP reporter were cultured in DMEM, supplemented with 10% FBS, 100 U/ml penicillin, 100 ng/ml streptomycin. 1 μg/ml of puromycin was added to the culture medium at all times. The generated stable cell clones were analyzed for GFP expression by flow cytometry every two weeks over a 4-month period. RR and RU cells were sorted out based on GFP expression and cultured separately. The two populations remained 98% pure over 4 months.
Gene silencing
MCF7 and ZR751 cells were transfected with 1 nmol of SMARTpool siRNA designed against Sox2 (Thermo Scientific). Scramble non-targeting siRNA (Thermo Scientific) was used as the negative control. For all siRNA transfection, a BTX 830 electroporation instrument (Harvard Apparatus, Holliston, MA) was used. For double knockdown experiments, SMARTpool siRNA designed against Twist1 from Thermo Scientific was used.
Enforced expression of Sox2 in MCF7 cells was performed as previously described [
39]. Briefly, pheonix packaging cells were transfected with either pMXs
Sox2 retroviral vector (Addgene, MA, USA) or empty vector according to the manufacturer's suggestion. MCF7 cells were infected with retroviral particles three times in 24 hour intervals. 48 hours after the final infection, cells were overnight starved and were then used to perform invasion assay.
Western blotting
Western blot analyses were performed as previously described [
40,
41]. The following antibodies were used: Sox2 (Cell Signaling Technologies), Twist1 (Santa Cruz), γ-Tubulin (Sigma).
Cell viability
Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay (Promega, Madison, WI) according to the manufacturer's protocol.
Cell invasion assay
As previously described, we assessed cell invasiveness using the Cytoselect™ 24-well cell invasion assay kit (Cell Biolabs, San Diego, CA, USA) according to the manufacture’ s protocol [
42]. Briefly, cells were overnight starved prior to invasion assay. Approximately 1 × 10
5 cells in serum free medium were plated in the top chamber and medium supplemented with 10% FBS was used as a chemo-attractant in the lower chamber. The cells were then allowed to invade the reconstituted basement membrane matrix for 24 hours. The invasive cells passed the membrane were then dissociated from membrane, lysed and quantified using CyQuant GR fluorescent Dye.
Quantitative RT-PCR
Total RNA was extracted using TRIzol according to the manufacturer’s protocol. Quantitative RT-PCR was performed using Applied Biosystem Prism 7900HT instruments. The TaqMan gene expression assay (Applied Biosystems) used were: Hs01548727_m1 (MMP2), Hs00234579_m1 (MMP9), Hs01675818_s1 (Twist1), Hs01023894_m1 (E-cadherin), Hs00362037_m1 (N-cadherin, Hs00232783_m1(ZEB1) and Hs00998133_m1 (TGF-β). Primer sequences for Snail are: Forward 5'-acaaaggctgacagactcactg-3′, Reward 5′-tgacagccattactcacagtcc-3′. Primer sequences for Slug are: Forward 5′-gtctctcctgcacaaacatgag-3′, Reverse 5′-atgctcttgcagctctctctct-3′. Primer sequences for MMP3: Forward 5′-cactcacagacctgactcggtt-3′, Reverse 5′- aagcaggatcacagttggctgg-3′. Primer sequence for FAK are Forward 5′-gccttatgacgaaatgctgggc-3′, Reverse 5′- cctgtcttctggactccatcct -3′. Human GAPDH was used as control. Expression of each gene was measured in triplicate.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was performed as our previously described [
39]. The chromatin was extracted from MCF7-RR and -RU cells. A normal rabbit IgG antibody and anti-Sox2 antibody (Santa Cruz) was then incubated with the chromatin. Isolated DNA was then amplified with
Twist1 primers (−1478 to −1322 of transcriptional start site, 156 bp amplicons): Forward 5′-ggcgagtccgtactgagaag-3′ Reverse 5′- cgtttcaggtccatccctta-3′.
Statistical analysis
All the statistical analyses were performed using the GraphPad Prism 5.1 program. Student T-test and One-way ANOVA were used to calculate p value. Results are presented as mean ± standard deviation.
Discussion
The aberrant expression of Sox2 in cancer cells has been found to correlate with the invasiveness of several types of solid tumors [
30,
34,
35,
37,
48‐
50]. For instance, a high level of Sox2 expression detectable by immunohistochemistry was found to correlate with higher invasiveness and metastatic potential in gliomas and colorectal cancer [
35,
36]. Furthermore, siRNA knockdown of Sox2 can result in decreased invasiveness in cell lines derived from gliomas, melanomas and colorectal cancer [
35‐
37]. However, it appears that Sox2 expression in cancer does not always correlate with increased invasiveness and metastasis. We found at least one previous study in which a relatively low level of Sox2 expression in gastric cancer correlates with increased invasiveness/metastatic potential [
34]. In the current study, we also found evidence that Sox2 suppresses invasiveness in BC. Thus, the biological effects of Sox2 in cancer cells are likely to be tumor type-specific.
Our finding that Sox2 suppresses the invasiveness of BC is in contrast with that made by another group, who found that enforced expression of Sox2 in MCF7 cells can increases their invasiveness by approximately 60% [
38]. In our study, we initially found that siRNA knockdown of Sox2 significantly increased the invasiveness of parental MCF7 cells and MCF7-RU cells. In view of the discrepancy between our conclusion and that described in the literature [
38], we attempted to replicate the experiment that examined the effects of enforced Sox2 over-expression in MCF7 cells, as described previously [
38], and we did not find any significant change in the invasiveness of these cells (Figure
1C). We would like to point out that the lack of response to enforced Sox2 expression in MCF7 is similar to the finding of one of previous studies, in which enforced expression of Sox2 in MCF7 cells was found to result in no significant change in mammosphere formation and cell growth [
39]. While we do not have definitive explanations for the discrepancy between our results and the previously published results [
38], we have considered the possibility that the MCF7 cell clones used in the two laboratories may be different. We also have considered the possibility that the
in-vitro invasiveness assays between the two laboratories have different characteristics. Lastly, since the exact Sox2 protein level has been shown to be functionally important in ESCs [
51,
52], it is possible that the total Sox2 protein levels after gene transfection are substantially different between the two laboratories, and thus, leading to substantially different biological responses.
The mechanisms by which Sox2 regulate tumor invasiveness have not been extensively studied. In the literature, we were able to identify only 3 studies that are directly relevant to this subject. In all of these three studies (using cell lines derived from colorectal cancer, melanomas and gliomas, respectively), siRNA knockdown of Sox2 was found to decrease invasiveness; in the same three studies, the decrease in invasiveness was found to correlate with a decreased expression level of one of the following molecules: MMP2, MMP3 or FAK [
36,
37,
53]. To our knowledge, the mechanisms by which Sox2 regulates invasiveness in BC are not known. Thus, we screened a panel of factors known to play roles in regulating cell invasiveness/EMT in various types of cancer. In contrast with the previous reports, we did not find any appreciable changes in the expression levels of MMP3, MMP2 and FAK. Instead, we identified Twist1 as the only protein that is regulated by Sox2 in RU cells.
Twist1 has been reported to be one of the master regulators of invasiveness and EMT, and dysregulation of Twist1 expression and function has been implicated to be associated with cancer progression [
54‐
56]. In BC, a high level of Twist1 expression is more common in invasive lobular carcinomas [
5]. While siRNA knockdown of Twist1 in BC cells led to a decrease in invasiveness [
57], enforced expression of Twist1 in BC cells converts its normal epithelial cell morphology to a spindle-like/fibroblastic morphology [
5,
58]. In keeping with the concept that Twist1 plays a key role in regulating invasiveness in BC, siRNA knockdown of Twist1 decreased the invasive-ness of both MCF7-RR and -RU cells by approximately 20-30% (Figures
4A and
5A).
As mentioned in the introduction, the expression of Twist1 has been shown to be regulated by a number of proteins such as STAT3, BMP2 and SRC-1. The expression of Sox2 has been shown to correlate with that of Twist1 in human glioblastoma cells [
59], although direct proof that Sox2 regulates the expression of Twist1 is lacking. For the first time, we have provided direct evidence that the expression of Twist1 in BC is regulated by Sox2, and this regulation only occurs in the RU cells. Results from our ChIP studies further support the fact that Twist1 is regulated by Sox2 only in RU cells. Although Sox2 does not respond to the reporter in RU cells, possibly due to the fact that Sox2 in RU cells cannot bind to the Sox2 binding motif present in the Sox2 reporter [
39], Sox2 in RU cells can bind to the alternative Sox2 binding motif present in the
Twist1 gene promoter and thus suppress its expression as well as invasiveness. These findings are in parallel to the findings that Sox2 is known to negatively regulate a set of genes in ESCs. In contrast, in RR cells, Sox2 does not bind to the promoter region of
Twist1 and the expression of Twist1 is regulated by other factors. The mechanism underlying the decision as to whether Sox2 binds to the
Twist1 gene promoter is under active investigation in our laboratory. Since the transcription activity of Sox2 in normal ESCs has been shown to be modulated by its binding partners, we speculated that a similar scenario may occur in BC cells. Taken together, our findings suggest that the Sox2 transcriptional activity and Twist1 can serve as markers to predict invasiveness in breast cancer cells.
An important concept emerged from the results of this study is related to the significance of the dichotomy of BC cells separated based on the differential responsiveness to the
Sox2 reporter. Specifically, based on our double siRNA knockdown experiments (Figure
4), the Sox2-Twist1 axis plays a key role in regulating the invasiveness in RU cells. In contrast, Twist1, but not Sox2, plays a key role in regulating the invasiveness of RR cells. While the true biological significance of these observations requires further studies, we believe that our results have highlighted a new level of biological complexity of BC. In view of this new knowledge, one may wonder if our current treatments of BC, which are designed based on the assumption that BC cells within a tumor are composed of a biologically uniform population of cancer cells, are fundamentally inadequate. This newly discovered biological complexity of BC cells may prompt us to consider treatment strategies that are based on the recognition of phenotypically distinct cell subsets in BC that are driven by different biochemical pathways.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
FW and XY performed experiments and analyzed data; PW, KJ, CW, DD, NK, YM and GB assisted with experiments; FW and RL designed the research plan; FW and RL wrote the manuscript. All authors’ read and approved the final manuscript.