We describe here an ordered, self-reinforcing, TGFβ-initiated signaling circuitry leading to EMT in mouse and human prostate cancer (C). TGFβ-dependent loss of KLF4 was sufficient to initiate Slug induction and EMT (). KLF4 and FOXA1 are epithelium-determining transcription factors that are shown here to directly suppress Slug transcription (). Slug appears to be the “gatekeeper” of EMT in this model, since depletion of Slug not only inhibits EMT but also inhibits expression of a variety of other EMT regulators such as Zeb2 and Snai1 (). In contrast, EMT occurs, although less efficiently, following Snai1 depletion. SLUG is expressed in normal prostate basal cells, suggesting that SLUG expression in tumor cells may represent a developmentally acquired competency. Finally, for selected E boxes in the promoters of the epithelial genes Klf4 and Foxa1, there exists SLUG-dependent PRC1/2 binding and transcriptional inhibition, strengthening continued Slug expression and commitment to the mesenchymal lineage ().
Importantly, the EMT circuitry discovered in the AC3 cell line appears relevant for human prostate cancer cell lines and clinical prostate cancers ( and ). In tissue arrays, decreased KLF4 and increased SLUG staining in primary prostate cancer relative to that seen with normal glands was observed in grade III and IV metastatic tumors that had demonstrated progression to metastasis. In addition, for individual samples, decreased KLF4 was significantly correlated with increased SLUG. The results of a recent gene-profiling meta-analysis and tissue array study concluding that KLF4 is downregulated in prostate cancer tissue with metastases support the KLF4 data presented here (33
TGFβ is a common inducer of EMT (37
). Although many changes in gene expression occur in association with EMT, the transcriptional mechanisms that lead to EMT initiation have not been clearly established (32
). We show that TGFβ treatment resulted in rapid loss of KLF4, leading to induction of Slug
, the EMT regulatory transcription factor. KLF4
depletion was sufficient to initiate Slug-
dependent EMT; in contrast, the inhibition of EMT occurred following stabilization of KLF4 protein secondary to inhibiting KLF4 ubiquitylation and proteosomal degradation (). Previous studies have demonstrated that TGFβ-dependent proteosomal degradation of KLF4 occurs in the absence of new protein synthesis, suggesting a direct effect (17
). Thus, KLF4 loss provides a mechanistic link between TGFβ action and the known EMT regulator SLUG.
TGFβ-dependent transcriptional inducers of Slug
have been described. In MDCK cells, TGFβ-initiated myocardin-related transcription factor (MRTF)-SMAD3 complex regulation was previously demonstrated (24
). In Panc1 cells, TGFβ induced SMAD3-IκB kinase 1 complex (SMAD3-IKK1) binding to the SLUG
promoter, and depletion of IKK1
). Taking these data together, TGFβ-initiated Slug
transcription appears to be regulated in a context-specific manner by repressors and inducers, at least some of which are dependent on the presence of SMAD. Interestingly, KLF4 has been shown to act as an SMAD corepressor in reporter assays (17
KLF4 is a pleiotropic regulator that influences growth, epithelial differentiation, and invasion in normal and tumor cells (20
). KLF4 can function as either an inductive or inhibitory transcription factor acting upon, for example, various cell cycle regulatory genes or differentiation genes, including CDH1
). Loss of KLF4 in various cancers, including colon, breast, and gastric cancers, is consistent with tumor-suppressive functions (3
). Ectopic overexpression of KLF4
inhibits growth and invasiveness of tumor cell lines, including prostate cancer (12
). The pleiotropic potential of KLF4 suggests that it may be an integrator of growth- and lineage-dependent differentiation functions.
Similarly to KLF4, FOXA1 is an epithelial determinant and, as shown here, functions as a direct transcriptional repressor of Slug.
In turn, Foxa1
expression is transcriptionally inhibited by increased SLUG binding, which occurs in combination with PRC1/2 complexes. In the prostate, FOXA1 is necessary for luminal ductal epithelial differentiation and functions as a required cofactor for androgen receptor binding to chromatin (15
). In addition, FOXA1 is a transcriptional inducer of CDH1
, a positive regulator of the epithelial phenotype (21
). Unlike those of Klf4, Foxa1 mRNA and protein levels decline gradually following TGFβ treatment, and Foxa1
downregulation is dependent on the presence of Slug
. Thus, loss of FOXA1 most likely plays a role in the later phases of transitioning to the mesenchymal state. These data further suggest that FOXA1 and KLF4 may functionally synergize in regulating partial or full EMT.
We anticipate that the EMT circuitry described here may be particularly relevant to TP53-deficient tumors. Deletion of TP53
is one of the most common copy number variations observed in human prostate cancer (31
). Basal and induced SLUG levels are expected to increase in TP53-deficient cells. TP53 induces SLUG degradation by upregulating MDM2 expression and participating in a TP53-MDM2-SLUG complex that facilitates MDM2-mediated SLUG degradation (34
). Loss of Tp53
in certain epithelial tumor models is associated with a propensity to form sarcomatoid carcinomas (8
In conclusion, we have used a genetically defined Pten/TP53 null mouse model of prostate cancer and human prostate cancer cells to show that EMT initiated by TGFβ proceeds by a self-reinforcing feedback loop between decreased KLF4 and increased SLUG levels. The data suggest that SLUG and KLF4 should be explored further as potential diagnostic and prognostic markers of invasive prostate cancer. We anticipate that this defined circuitry will provide insight for investigating the regulation of EMT phenotypes that are proposed to function during tumor progression.