Lack of an Additive Effect between the Deletions of Klf5 and Nkx3-1 in Mouse Prostatic Tumorigenesis

Prostate cancer is one of the most common malignancies and a leading cause of cancer death in industrialized countries. The development and progression of prostate cancer are driven by a series of genetic and epigenetic events including gene amplification that activates oncogenes and chromosomal deletion that inactivates tumor suppressor genes. Whereas gene amplification occurs in human prostate cancer, gene deletion is more common, and a large number of chromosomal regions have been identified to have frequent deletion in prostate cancer, suggesting that tumor suppressor inactivation is more common than oncogene activation in prostatic carcinogenesis (Knuutila et al., 1998, 1999; Dong, 2001). Among the most frequently deleted chromosomal regions in prostate cancer, target genes such as NKX3-1 from 8p21, PTEN from 10q23 and ATBF1 from 16q22 have been identified by different approaches (He et al., 1997; Li et al., 1997; Sun et al., 2005), and deletion of these genes in mouse prostates has been demonstrated to induce and/or promote prostatic carcinogenesis. For example, knockout of Nkx3-1 in mice induces hyperplasia and dysplasia (Bhatia-Gaur et al., 1999; Abdulkadir et al., 2002) and promotes prostatic tumorigenesis (Abate-Shen et al., 2003), while knockout of Pten alone causes prostatic neoplasia (Wang et al., 2003). Therefore, gene deletion plays a causal role in prostatic carcinogenesis (Dong, 2001).

The deletion of the q21 region of human chromosome 13 (13q21) is the second most frequent deletion in human prostate cancer and other types of malignancies (Knuutila et al., 1999; Dong et al., 2000; Chen et al., 2001; Dong, 2001), and the KLF5 gene has been suggested to be the 13q21 tumor suppressor gene because it centers the 13q21 deletion (Chen et al., 2003). A tumor suppressor activity has been detected for KLF5 in mouse models, including xenograft models in which KLF5 expression inhibits the tumorigenesis of human prostate cancer cell lines (Nakajima et al., 2011)and the knockout mouse model in which knockout of Klf5 increases cell proliferation and induces hyperplasia in prostate epithelial cells (data not shown), and promotes the effect of Pten deletion in prostatic carcinogenesis (data not shown).

In human prostate cancer, genomic deletion often involves simultaneous deletions of multiple chromosomal regions, and the two most common regions of deletion are 8p21 and 13q21 (Knuutila et al., 1999; Dong, 2001). For example, among six prostate cancer cell lines examined for chromosomal deletion by comparative genomic hybridization, three (NCI-H660, PC-3 and PPC-1) showed deletions of both 8p and 13q (Pan et al., 2001). It is thus expected that simultaneous deletions of multiple regions and genes are necessary for a tumor cell to arise. It has been demonstrated in mouse models that deletion of one gene is often insufficient for tumor induction, and deletion of two or more genes has an additive or synergistic effect on tumorigenesis. For example, deletion of both Pten and Nkx3-1 in mouse prostates causes invasive prostate cancer and metastasis while the deletion of either one alone has a much milder effect (Bhatia-Gaur et al., 1999; Abdulkadir et al., 2002; Abate-Shen et al., 2003; Wang et al., 2003; Shappell et al., 2004). Similarly, while the deletion of Klf5 alone causes hyperplasia in mouse prostates (data not shown), it significantly promotes the oncogenic effect of Pten deletion (data not shown).

In this study, we tested whether the two most frequent genetic deletions in human prostate cancer, NKX3-1 at 8p21 and KLF5 at 13q21, have any additive or synergistic effects on prostatic tumorigenesis. We bred mouse strains PB-Cre4, Flox-Klf5 and Nkx3-1 null(−/−) to generate the following six desired genotypes: wild type (wt), hemizygous deletion and homozygous deletion of Klf5 with both heterozygous(+/−)Nkx3-1 (Cre⁺/Klf5^wt/wt/Nkx3-1^+/−, Cre⁺/Klf5^flox/wt/Nkx3-1^+/− and Cre⁺/Klf5^flox/flox/Nkx3-1^+/−, respectively) and null Nkx3-1(Cre⁺/Klf5^wt/wt/Nkx3-1^−/−, Cre⁺/Klf5^flox/wt/Nkx3-1^−/−, and Cre⁺/Klf5^flox/flox/Nkx3-1^−/− respectively) (Fig. S1A). At the ages of 18 months for the Nkx3-1heterozygous group and one year for the Nkx3-1 null group, mice were sacrificed and the prostates were immediately isolated for histopathological and molecular analyses. The efficiency of knockout was confirmed by immunohistochemistry (IHC) or immunofluorescent (IF) staining in the anterior prostate (Fig. S1B, C), where an obvious decrease in protein expression indicates the successful knockout of that gene in the prostate.

Previous studies indicate that in dorsal-lateral and anterior prostates, both the Klf5 knockout and Nkx3-1 knockout induce phenotypic alterations. Knockout of Klf5 alone increases cell proliferation and causes hyperplasia when it occurs at one Klf5 allele, but causes cell death when it occurs at both alleles (data not shown). Knockout of Nkx3-1, on the other hand, results in epithelial hyperplasia and dysplasia in the prostate regardless of the number of alleles that is knocked out (Bhatia-Gaur et al., 1999). We found that when one allele of Nkx3-1 and either one or both alleles of Klf5 were knocked out, only the Nkx3-1 phenotype, i.e., epithelial hyperplasia and dysplasia (Bhatia-Gaur et al., 1999), was detectable in the prostate (Figs. 1 and S2). The deletion of Klf5, at either one or both alleles, did not result in any additional detectable effects on the prostate, including the size and structure of acini, the thickness of epithelia (layers of cells), and the number of glandular in foldings (Figs. 1 and S2). No prostatic intraepithelial neoplasia (PIN), a well established neoplastic alteration in mouse prostates, was detected in these mice. We further found no difference in the knockout of one or both Klf5 alleles, even though they had different effects on prostatic epithelia when without the knockout of other genes (data not shown).

Fig. 1. Histological analysis and proliferation detection of mouse prostates with the knockout of Klf5 in Nkx3-1 knockout mice.

A:Nkx3-1^+/− and Nkx3-1^−/− mice of three Klf5 genotypes (Klf5^+/+, Klf5^+/− and Klf5^−/−) were sacrificed at the ages of 18 months and one year, respectively, and tissues were subjected to hematoxylin and eosin (HE) staining and histopathological analysis. Shown are dorsal-lateral prostates (DLP) and anterior prostates (AP) with 50× magnification. The frame in each figure indicates the exact region shown in Fig. S2, in which 200× magnification is chosen. B:Ki67, a proliferating marker, was stained by IHC staining in DLP and AP from 18-month old mice with the knockout of Klf5 and one allele of Nkx3-1 or from one-year old mice with the knockout of Klf5 and both alleles of Nkx3-1.

When both Nkx3-1 alleles were knocked out, more severe epithelial hyperplasia and dysplasia were observed as expected (Bhatia-Gaur et al., 1999), yet the deletion of Klf5 again did not cause additional phenotypic alterations, regardless of the number of Klf5 alleles that were deleted (Figs. 1 and S2). No PIN was observed in the prostate when both Nkx3-1 alleles were knocked out.

We also measured the rate of cell proliferation by analyzing the number of cells that expressed Ki67, a marker for cell proliferation, in the prostate by IHC staining and cell counting. To countKi67-expressing cells and total epithelial cells, four randomly selected fields per genotype were used. The percentages of Ki67-positive cells were calculated (Table S1). Whereas the knockout of Nkx3-1 alone, at one or both alleles, significantly increased the rate of Ki67-expressing cells as expected (Bhatia-Gaur et al., 1999), knockout of Klf5, at either one or both alleles, did not cause an additional increase (Table S1). This result is consistent with the findings in phenotypic alteration (Fig. 1), suggesting that the Nkx3-1 knockout and the Klf5 knockout do not have an additive effect.

In the ventral prostate, which has no anatomical counterpart in human prostate (Valkenburg and Williams, 2011), neither Nkx3-1 deletion nor Klf5 deletion had any detectable effect on the morphology (data not shown).

Different genetic alterations such as deletions of NKX3-1 at 8p21, KLF5 at 13q21 and PTEN at 10q23, all of which frequently occur in human prostate cancer, may interact with an additive or synergistic effect or may function without any interactions. Although both interact with the deletion of Pten to promote prostatic carcinogenesis (Abate-Shen et al., 2003;), the deletion of Nkx3-1 and deletion of Klf5 did not have an additive effect on prostatic carcinogenesis, as indicated by cell proliferation rate and prostate morphology (Fig. 1 and Table S1).

There are at least two possible explanations for the lack of interaction between Klf5 deletion and Nkx3-1 deletion. The first is that the effect of either Klf5 deletion orNkx3-1 deletion on the prostate is much milder than that of Pten deletion, as Nkx3-1 deletion induces hyperplasia and dysplasia and Klf5 deletion induces hyperplasia, but Pten deletion causes the more severe PIN (Bhatia-Gaur et al., 1999; Wang et al., 2003). It is thus possible that the combination of two milder lesions is still not sufficient for the induction of PIN and prostate tumorigenesis. The second explanation is that inactivation of Nkx3-1 or Klf5 functions as a secondary event in the development of prostate cancer, and without a major event such as Pten inactivation, these secondary events are insufficient for any more severe alterations regardless of how many such events occur simultaneously.

Compared to the effect of Pten deletion alone, both the Pten-Nkx3-1 knockout and the Pten-Klf5 knockout caused larger tumors, further increased cell proliferation, and induced more severe phenotypes in the dorsal-lateral prostates than in the anterior and ventral prostates (Abate-Shen et al., 2003). In addition, the smooth muscle layer surrounding the epithelium was thinner and discontinuous in both double knockout groups. These similarities suggest that Klf5 deletion and Nkx3-1 deletion may have a similar effect on prostatic tumorigenesis. Nkx3-1 and the Myc oncoprotein cross-regulate target genes in mouse and human prostate tumors (Anderson et al., 2012), and Myc overexpression causes PIN accompanied by the loss of Nkx3-1 (Iwata et al., 2010). On the other hand, KLF5 directly regulates the expression of Myc in epithelial cells (Guo et al., 2009). It is thus also possible that KLF5 and NKX3-1 function on the same molecular pathway in the regulation of cell proliferation, differentiation and tumorigenesis.

In summary, we examined whether deletion of Klf5 and deletion of Nkx3-1, the two most common genomic deletions in human prostate cancer, have additive and/or synergistic effects on prostatic carcinogenesis. Analysis of prostate morphology and cell proliferation showed that these two genetic events do not have a detectable interaction in prostatic carcinogenesis. Different mechanisms could be responsible for this lack of interaction.