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
. 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%
. 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
, we attempted to replicate the experiment that examined the effects of enforced Sox2 over-expression in MCF7 cells, as described previously
, 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
. While we do not have definitive explanations for the discrepancy between our results and the previously published results
, 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
. While siRNA knockdown of Twist1 in BC cells led to a decrease in invasiveness
, 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
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
, 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
, 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.