The Krüppel-like factor 4 (KLF4) is a transcriptional regulator of proliferation and differentiation in epithelial cells, both during development and tumorigenesis. Although KLF4 functions as a tumor suppressor in several tissues, including the colon, the role of KLF4 in breast cancer is less clear. Here, we show that KLF4 is necessary for maintenance of the epithelial phenotype in non-transformed MCF-10A mammary epithelial cells. KLF4 silencing led to alterations in epithelial cell morphology and migration, indicative of an epithelial-to-mesenchymal transition. Consistent with these changes, decreased levels of KLF4 also resulted in the loss of E-cadherin protein and mRNA. Promoter/reporter analyses revealed decreased E-cadherin promoter activity with KLF4 silencing, while chromatin immunoprecipitation identified endogenous KLF4 binding to the GC-rich/E-box region of this promoter. Furthermore, forced expression of KLF4 in the highly metastatic MDA-MB-231 breast tumor cell line was sufficient to restore E-cadherin expression and suppress migration and invasion. These findings identify E-cadherin as a novel transcriptional target of KLF4. The clear requirement for KLF4 to maintain E-cadherin expression and prevent epithelial-to-mesenchymal transition in mammary epithelial cells supports a metastasis suppressive role for KLF4 in breast cancer.Krüppel-like factor 4 (KLF4) 3 is a zinc finger transcription factor that was first identified in a screen for transcription factors involved in growth regulation (1). KLF4 is primarily regarded as a negative regulator of the cell cycle, repressing a multitude of genes that promote proliferation while at the same time up-regulating inhibitors of proliferation (2). KLF4 also plays a crucial role in differentiation during organogenesis of various tissues such as the skin, colon, and eye (3-5). With the advent of induced pluripotent stem cells, KLF4 has gained recognition as one of the "pluripotency genes" that can reprogram somatic cells into a stem cell-like state (6), acting in the capacity to maintain self-renewal (7).Given its stem cell-promoting activity and its ability to regulate growth and differentiation during development, it is not surprising that KLF4 also plays various roles in tumorigenesis. The frequent loss of KLF4 expression in gastric and colorectal cancers has led to studies revealing a tumor-suppressive role for this factor in these and other tissues (8 -13). Conversely, overexpression of KLF4 in the skin leads to squamous cell carcinoma (14). However, the role of KLF4 during the progression of breast cancer is not well defined. Immunohistochemical studies have revealed that KLF4 expression can be increased and undergo altered localization in DCIS of the breast (15), suggesting that it may act as an oncogene in this tissue. This is further supported by the association of nuclear KLF4 with an aggressive breast cancer phenotype (16). In contrast, Akaogi et al. reported that a review of nine independent, publicly available gene expression data sets revealed de...

Krüppel-like factor 4 (KLF4) is a zinc finger transcription factor that functions as an oncogene or tumor suppressor in a highly tissue-specific cell-dependent manner. However, its precise role in breast cancer and metastasis remains unclear. Here, we show that transient adenoviral expression of KLF4 in the 4T1 orthotopic mammary cancer model significantly attenuated primary tumor growth as well as micrometastases to the lungs and liver. These results can be attributed, in part, to decreased proliferation and increased apoptosis. Further supporting a tumor-suppressive role for KLF4 in the breast, we found that KLF4 expression is lost in a mouse model of HER2/NEU/ERBB2-positive breast cancer. To determine whether enforced KLF4 expression could alter tumor latency in these mice, we used a doxycycline-inducible expression model in the context of the MMTV-Neu transgene. Surprisingly, tumors that developed in this model also lost KLF4 expression, suggesting negative selection for sustained expression. We have previously reported that KLF4 inhibits epithelial-to-mesenchymal transition (EMT), a preliminary step in metastatic progression. Overexpression of KLF4 in 4T1 cells led to a significant reduction in the expression of Snail, a key mediator of EMT and metastasis. Conversely, KLF4 silencing increased Snail expression in the nontransformed MCF-10A cell line. Collectively, these data demonstrate the first functional, in vivo evidence for KLF4 as a tumor suppressor in breast cancer cells. Furthermore, our findings suggest an inhibitory role for KLF4 during breast cancer metastases that functions, in part, through repression of Snail.

Breast cancers that overexpress the receptor tyrosine kinase ErbB2/HER2/Neu result in poor patient outcome because of extensive metastatic progression. Herein, we delineate a molecular mechanism that may govern this malignant phenotype. ErbB2 induction of migration requires activation of the small GTPases Rac1 and Cdc42. The ability of ErbB2 to activate these small GTPases necessitated expression of p120 catenin, which is itself up-regulated by signaling through ErbB2 and the tyrosine kinase Src. Silencing p120 in ErbB2-dependent breast cancer cell lines dramatically inhibited migration and invasion as well as activation of Rac1 and Cdc42. In contrast, overexpression of constitutively active mutants of these GTPases reversed the effects of p120 silencing. Lastly, ectopic expression of p120 promoted migration and invasion and potentiated metastatic progression of a weakly metastatic, ErbB2-dependent breast cancer cell line. These results suggest that p120 acts as an obligate intermediate between ErbB2 and Rac1/Cdc42 to modulate the metastatic potential of breast cancer cells.HER2/Neu/ErbB2 (epidermal growth factor receptor 2) is a member of the epidermal growth factor receptor (EGFR) 2 family that is amplified and overexpressed in 20 -30% of breast cancers. HER2/ErbB2-positive breast cancer yields a poor patient prognosis because of a high incidence of metastases and intrinsic resistance to endocrine and conventional chemotherapy (1). ErbB2 is the preferred heterodimerization partner for EGFR, ErbB3, and ErbB4 (2). The large range of downstream signaling targets induced by ErbB2-containing heterodimers permits this receptor and its partners to modulate a wide array of cellular processes. Of these, the nonreceptor tyrosine kinase Src, PI3K, and MAPK pathways are the most studied in mediating ErbB2 responsiveness, which includes increased proliferation, survival, motility, and invasion (3).ErbB2-induced cell migration and invasion, and hence metastatic potential, requires activation of Src and its downstream signaling pathways (4). Src also positively provides feedback and stabilizes competent ErbB2/ErbB3 heterocomplexes to enhance PI3K/Akt signaling (5). ErbB2 overexpression in MDA-MB-435 cells enhances formation of metastases by increasing Src synthesis and decreasing its degradation by calpain (6). Similarly, signaling from ErbB2 to Akt is necessary to promote metastatic potential because attenuation of the Akt pathway by pharmacological inhibitors or RNAi decreases the formation of metastases by the ErbB2-dependent 21T breast cancer cell line (7).

Cadherin cell-cell adhesion proteins play an important role in modulating the behavior of tumor cells. E-cadherin serves as a suppressor of tumor cell invasion, and when tumor cells turn on the expression of a non-epithelial cadherin, they often express less E-cadherin, enhancing the tumorigenic phenotype of the cells. Here, we show that when A431 cells are forced to express R-cadherin, they dramatically downregulate the expression of endogenous E-and P-cadherin. In addition, we show that this downregulation is owing to increased turnover of the endogenous cadherins via clathrin-dependent endocytosis. p120 ctn binds to the juxtamembrane domain of classical cadherins and has been proposed to regulate cadherin adhesive activity. One way p120 ctn may accomplish this is to serve as a rheostat to regulate the levels of cadherin. Here, we show that the degradation of E-cadherin in response to expression of R-cadherin is owing to competition for p120 ctn .

Classical cadherins are the transmembrane proteins of the adherens junction and mediate cell-cell adhesion via homotypic interactions in the extracellular space. In addition, they mediate connections to the cytoskeleton by means of their association with catenins. Decreased cadherin-mediated adhesion has been implicated as an important component of tumorigenesis. Cadherin switching is central to the epithelial to mesenchymal transitions that drive normal developmental processes. Cadherin switching has also been implicated in tumorigenesis, particularly in metastasis. Recently, cadherins have been shown to be engaged in cellular activities other than adhesion, including motility, invasion, and signaling. In this study, we show that inappropriate expression of R-cadherin in tumor cells results in decreased expression of endogenous cadherins (cadherin switching) and sustained signaling through Rho GTPases. In addition, we show that R-cadherin induces cell motility when expressed in epithelial cells and that this increased motility is dependent upon Rho GTPase activity.Classical cadherins are the transmembrane components of the adherens junction. E-Cadherin is found in epithelial cell junctions (E-cadherin), 1 whereas other cadherins are found in similar structures in other cell types (1, 2). Disruption of Ecadherin function through mutations in E-cadherin itself, through modifications to the E-cadherin promoter or through disruption of the cadherin/catenin complex, contributes to the aggressive behavior of epithelial-derived tumor cells (1). Studies from our laboratory and others have shown that expression of an inappropriate cadherin in epithelial cells is another way that tumor cells can alter their adhesive function (3-6). In some cases, this is due to down-regulation of E-cadherin upon expression of the inappropriate cadherin (4). In other cases, neuronal cadherin (N-cadherin) can have a direct and dominant influence upon the phenotype of epithelial cells, despite their continued expression of E-cadherin. For example, expression of N-cadherin by oral squamous epithelial cells results in decreased expression of the endogenous cadherins, whereas expression of N-cadherin by some breast epithelial cells has no effect upon expression of endogenous cadherins, but none the less influences cell adhesion and behavior (4, 5). Thus, exogenous expression of N-cadherin can modulate the expression of endogenous cadherins in some cell types but not in others. Interestingly, exogenous expression of vascular endothelial cadherin (VE-cadherin) in a variety of cell types does not influence the expression levels of any endogenous cadherins (7). However, VE-cadherin does prevent proper junctional localization of N-cadherin, but has no influence upon the localization of other cadherins (7). In the present study, we show that exogenous expression of retinal cadherin (R-cadherin) down-regulates the expression of all endogenous cadherins in every cell type examined. Thus, it is clear that cells have a mechanism for sensing cadherin exp...