The role of KLF4 in prostate cancer has never been investigated. Within this study, we reveal that KLF4 is downregulated in prostate cancer cell lines and prostate cancer tissue with metastases. Interestingly, all 6 prostate cancer cell lines evaluated in this study, with exception to RV1, are derived from metastatic prostate tissue (32
). Analysis of 47 total datasets from the Oncomine database revealed that no dataset recorded KLF4 upregulation in diseased tissue. Rather, the only significant changes in KLF4 expression were downregulation with increased frequency in metastatic sample datasets. Analysis of patient-matched tissues also revealed that low KLF4 immunostaining may have prognostic value for prostate cancer metastasis. Collectively, this data suggests that KLF4 functions as an inhibitor of prostate cancer progression. In support, restoration of KLF4 expression significantly reduced prostate cancer cell viability, clonogenicity, growth, migration, and invasion – features indicative of tumor suppressor function. Such phenotypes also associated with changes in gene expression known to negatively impact cell growth and migration. For instance, KLF4 induced p27 expression in prostate cancer cell lines; low p27 levels associate with aggressive/recurrent prostate cancer, while its overexpression correlates with growth inhibition in cell culture (34
KLF4 has also been shown to inhibit migration and invasion in several cancer models (3
). Interestingly, forced expression of KLF4 in prostate cancer cells elevated expression of TIMP2; a metalloproteinase thought to function as an inhibitor of metastasis. TIMP2 has been reported to be inactivated via
DNA hypermethylation in metastatic prostate cancer tissue and inhibit cancer cell migration (37
). In this regards, TIMP2 may function as a downstream factor of KLF4 to inhibit cell migration/invasion in prostate cancer.
RNAa-mediated overexpression of KLF4 inhibited cell proliferation/survival and arrested cell cycle progression. In DuPro cells, KLF4 overexpression resulted in G2/M arrest, while accumulation in G1/G0 phases of the cell cycle was detected in PC-3 cells. KLF4 has been shown to cause either G1 or G2 arrest in different cell types based on modulation and function of downstream genes (10
). Differential regulation of p57 protein levels between PC-3 and DuPro cells may play a role in delineating alternate phases of cell cycle arrest; elevated levels of p57 associate with G1 arrest (40
). Declines in CCNB1 may contribute to G2 arrest as KLF4 has been previously shown to prevent entry into mitosis through reductions in CCNB1 levels (10
). Based on combination and susceptibility of downstream genes to KLF4 reactivation, DuPro and PC-3 cells were inhibited at different phases of the cell cycle. The ability of KLF4 to inhibit cell cycle progression at multiple checkpoints is indicative of a gene with potent tumor suppressor-like function. Transfection of dsKLF4-496 also led to a small population of cells with sub-diploid DNA content in both DuPro and PC-3 cells. Enhanced cell death may also contribute to the tumor suppressor-like activity of KLF4 in prostate cancer cells.
Vector-based overexpression is the traditional approach to evaluate the function of tumor suppressor genes or oncogenes in cancer cells. However, all vector-based systems require ectopic expression from an exogenous construct. Ectopic expression vectors do not typically resemble natural genes (41
). They are frequently created from cDNA libraries or amplicons, which do not contain introns or UTR elements. Such regions can have pivotal effects on endogenous gene function in cancer biology (41
). As a laboratory tool, RNAa has the unique ability to enhance endogenous transcription of a targeted gene. Such a technique may allow for a more natural approach at analyzing gene function. In support, RNAa appeared to facilitate a greater measurable effect on downstream gene expression compared to viral transduction ( and Fig. S6
In conclusion, both cDNA and protein expression profiling demonstrated that KLF4 is significantly downregulated in prostate cancer tissue with metastases. Furthermore, decreased KLF4 expression has potential to serve as a strong and independent predictor of prostate cancer metastasis. By using RNAa as a laboratory technique, we revealed that KLF4 inhibits prostate cancer cell growth/survival and arrests cell cycle progression via modulating the expression of key downstream genes. KLF4 also suppressed prostate cell migration/invasion and upregulated TIMP2. This study reveals that KLF4 functions as a putative tumor suppressor in prostate cancer, as well as demonstrates the applicability of RNAa to study gene function. Much like RNAi, RNAa offers a new therapeutic approach for combating disease at the genetic level. As such, the ability to re-activate KLF4 by RNAa may have therapeutic potential in the treatment of metastatic prostate cancer.