Although earlier reports have shown that 70% of breast carcinomas have elevated KLF4 expression and that increased nuclear staining for KLF4 is associated with a more aggressive phenotype (Foster et al., 2000
; Pandya et al., 2004
), the ability of KLF4 to initiate aggressive tumors in animal models has not been examined in vivo
. By performing a colony formation assay and using a xenograft tumor model, we confirmed that KLF4 knockdown inhibited mammary tumor development in vitro
and in vivo
(), suggesting that KLF4 could act as an oncogenic protein in breast cancers.
Earlier studies showed that the anti-proliferative function of KLF4 is associated with inhibition of cyclin-D1 (Shie et al., 2000
) and activation of the cell-cycle inhibitor p21CIP1
(Zhang et al., 2000
). However, inactivation of either protein not only neutralizes the cytostatic effect of KLF4, but also collaborates with KLF4 in oncogenic function by an in vitro
study (Rowland et al., 2005
), thus further highlighting the importance of p21CIP1
. Although this study provided a possible mechanistic explanation for the context-dependent oncogenic or tumor-suppressor functions of KLF4, it has not been validated in vivo
. In addition, a cellular mechanism by which KLF4 contributes to the aggressive characteristics of breast cancers remains elusive. Our current studies indicate that KLF4 is required for the maintenance of breast cancer stem cells (), and KLF4 knockdown significantly delayed tumor development of breast cancer cells in a xenograft mouse model (). In our studies, we provided data demonstrating that knockdown of KLF4 significantly decreased CSC-enriched populations by using several different CSC markers. It should be noted that we cannot exclude other explanations of our results. As we mentioned before, KLF4 exerted an anti-apoptotic function in many cancer cell lines. It is possible that the reduced CSC population in KLF4 knockdown cells may be a result of the increased apoptosis mediated by KLF4 reduction. However, the fact that cell viability of KLF4 knockdown cells was comparable to that of the control cells would argue against this possibility (data not shown). In addition, we have not performed limiting-dilution assays (LDAs) to determine the tumor-initiating capacities of CSC cells in NOD/SCID mice, which is a traditional method in CSC studies. However, the fact that stem cell properties in KLF4 knockdown cells were characterized by specific cell surface markers, side population, and aldehyde dehydrogenase, importance of which has widely been confirmed in tumorigenecity, and that KLF4 knockdown cells showed decreased tumorigenesis in NOD/SCID mice would still support an important role of KLF4 in maintenance of breast cancer stem cells. Nevertheless, our results not only provide additional experimental support for the important function of KLF4 in stem cell biology, as shown before in embryonic stem cells (Li et al., 2005
) and in iPS cells (Takahashi and Yamanaka, 2006
; Wernig et al., 2007
), but also are important for breast cancer studies. Cancer stem cells have been shown to foster blood vessel formation and promote cell motility. They have marked therapeutic resistance (Charafe-Jauffret et al., 2008
) and have been implicated in breast cancer metastasis that remains the number one cause of cancer-related mortality in women (Lawson et al., 2009
). Our study suggested that overexpression of KLF4 was sufficient to drive cell migration and invasion (). Additional studies on the mechanisms by which KLF4 maintains cancer stem cell phenotype will be very helpful to develop novel therapeutic strategies targeting KLF4 or the related signaling pathway to treat malignant breast cancer and metastasis.
The function of KLF4 in maintenance of cancer stem cells has been confirmed in our study by using Kenpaullone, a small molecule inhibitor of KLF4. Previous work has demonstrated that Kenpaullone is able to replace KLF4 in the reprogramming of primary and secondary fibroblasts and Kenpaullone-induced iPS cells display characteristics of pluripotent ES cells (Lyssiotis et al., 2009
). However, no difference of KLF4 expression has been observed after Kenpaullone treatment. Our study indicated that Kenpaullone treatment led to decreased expression of KLF4 both at the mRNA and protein levels (). This discrepancy may have arisen from two different systems, the previous one dealing with iPS cells and ours with breast cancer cells. More importantly, we postulate that KLF4 may be an early responsive gene after Kenpaullone treatment. In the iPS studies, KLF4 expression was detected after 5-day Kenpaullone incubation. The early responsiveness of KLF4 to Kenpaullone may have been missed. In our system, a maximal down-regulation of KLF4 was observed at a 4 h time point after Kenpaullone treatment (), after which its expression gradually recovered. Nevertheless, Kenpaullone-treated cells possessed phenotypes similar to KLF4 knockdown cells in our studies, which, from another point of view, confirmed the indispensable role of KLF4 in cancer stem cells and extended a function of Kenpaullone from the induction of iPS cells to the maintenance of mammary cancer stem cells.
The epithelial-mesenchymal transition (EMT), as a unique process by which epithelial cells undergo remarkable morphological changes (leading to increased motility and invasion), is believed to be reminiscent of “cancer stem-like cells”, showing characteristics similar to many cancer systems (Mani et al., 2008
; Yang and Weinberg, 2008
). TGF-β is a well established regulator of EMT during development and tumor progression (Miettinen et al., 1994
). King et al.
observed that TGF-β induced KLF4 synthesis in vascular smooth muscle cells (King et al., 2003
). In addition, β-catenin, one of the most important mesenchymal markers, has been verified to interact with KLF4 in the intestinal crypts, with the crosstalk of KLF4 and β-catenin playing a critical role in the homeostasis of the normal intestine as well as in the tumorigenesis of colorectal cancers (Zhang et al., 2006
). Based on the pivotal role of KLF4 in cancer stem cells, in combination with its links with TGF-β signaling pathway, we highly suspected that KLF4 promoted EMT in breast cancers. In our studies, KLF4 knockdown MCF-7 cells exhibited a well-spread morphology, with the majority of cells forming a rounded, epithelial-like form and aggregating together in groups, a typical characteristic of MET and a reversal of EMT (data not shown). In addition, two critical mesenchymal-associated markers, fibronectin and vimentin, were decreased in siKLF4 (MCF-7 and MDA-MB-231) cells (data not shown), which were consistent with reduced ability of migration and invasion of siKLF4 cells. However, E-cadherin expression and localization, a hallmark of the EMT phenotype, showed no significant difference between siCon and siKLF4 cells. Recently, KLF4 was reported to inhibit EMT in non-transformed MCF-10A cells (Yori et al.
), which was entirely the opposite of our results. Our major argument is that MCF-10A cells are spontaneously transformed cells with no potential of tumorigenesis. Therefore, the results from MCF-10A cells may not be readily applicable to other mammary tumor cells. In their study, MDA-MB-231 tumor cells with KLF4 overexpression had also been used. However, results from our studies, using KLF4 knockdown and overexpression stable cells, supported a positive connection between KLF4 and EMT. Clearly, more studies are necessary to examine whether the difference of the two systems or the genetic background of specific MDA-MB-231 clones contributes to the discrepancies between the previously reported results and our current results.
It has been reported that Notch signaling plays a critical role in normal human mammary development by acting on both stem cells and progenitor cells (Dontu et al., 2004
), suggesting that abnormal Notch signaling may contribute to mammary carcinogenesis by deregulating the self-renewal of normal mammary stem cells. In this current study, we found that the expression of Notch1, Notch2 and Jagged1 was significantly decreased in KLF4 knockdown cells (). Unexpectedly, inhibition of the Notch pathway by a GSI had no effect on stem cell numbers and self-renewal potential of breast cancer cells (), suggesting that the Notch signaling pathway is not required for KLF4-mediated maintenance of stem cells in breast cancer cells. On the other hand, inhibition of Notch signaling by CompE in KLF4-overexpressing cells led to decreased migration and invasion ability (), which indicated that the Notch signaling pathway was responsible for KLF4-mediated mobility characteristics of breast cancer cells. These results are consistent with the role of Notch signaling as potent drivers during tumor progression and in converting polarized epithelial cells into motile, invasive cells (Sahlgren et al., 2008
). The relationship between the Notch signaling pathway and KLF4 appears dependent on different cellular contexts. Our early work and that of others suggest that KLF4 is inhibited by Notch in the gastrointestinal tract (Ghaleb et al., 2008
; Real et al., 2009
; Zheng et al., 2009
). Recently, downregulation of Notch1 gene expression in keratinocytes by KLF4 has also been reported (Lambertini et al.
). However, in our current studies, it appears that KLF4 upregulates gene expression of Notch1, Notch2, and Jagged1 (). This regulation likely occurs at the transcriptional level, as recently reported (Liu et al., 2009
), though an indirect effect cannot be ruled out.
In conclusion, our study provides evidence for the first time showing that KLF4 is essential for the maintenance of breast cancer stem cells and cell migration and invasion, which may offer important clues to understand how KLF4 promotes breast cancer development. Additional studies on the underlying mechanism will be very helpful to develop KLF4-based therapeutic strategies to treat breast cancer. In addition, whether KLF4 has the similar functions to maintain other cancer stem cells and to facilitate cell motility remains a topic for further investigation.