LZTS2 and PTEN collaboratively regulate ß-catenin in prostatic tumorigenesis

The leucine zipper tumor suppressor 2 (LZTS2) was identified as a tumor susceptibility gene within the 10q24.3 chromosomal region, and is approximately 15Mb from the PTEN locus. This region containing the both loci is frequently deleted in a variety of human malignancies, including prostate cancer. LZTS2 is a ß-catenin-binding protein and a negative regulator of Wnt signaling. Overexpression of PTEN in prostate cancer cell lines reduces ß-catenin-mediated transcriptional activity. In this study, we examined the collaborative effect of PTEN and LZTS2 using multiple in vitro and in vivo approaches. Co-expression of PTEN and LZTS2 in prostate cancer cells shows stronger repressive effect on ß-catenin mediated transcription. Using a newly generated mouse model, we further assessed the effect of simultaneous deletion of Pten and Lzts2 in the murine prostate. We observed that mice with both Lzts2 and Pten deletion have an earlier onset of prostate carcinomas as well as an accelerated tumor progression compared to mice with Pten or Lzts2 deletion alone. Immunohistochemical analyses show that atypical and tumor cells from compound mice with both Pten and Lzts2 deletion are mainly composed of prostate luminal epithelial cells and possess higher levels of cytoplasmic and nuclear β-catenin. These cells also exhibit a higher proliferative capacity than cells isolated from single deletion mice. These data demonstrate the significance of simultaneous Pten and Lzts2 deletion in oncogenic transformation in prostate cells and implicates a new mechanism for the dysregulation of Wnt/β-catenin signaling in prostate tumorigenesis.


Introduction
The leucine zipper tumor suppressor 2 (LZTS2), also called Lapser1, was originally identified based on homology with the LZTS1 tumor suppressor [1]. Lzts2 null mice showed no obvious pre-or post-natal lethality, but a portion of the mice developed defects in the kidney and urinary tract, including renal/ureteral duplication, hydroureter, and hydronephrosis [2]. Aged Lzts2 null mice also presented with increased spontaneous tumor development [3]. When treated with N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN), both homozygous and heterozygous Lzts2 deletion mice showed increased susceptibility to urinary bladder carcinoma development [3]. LZTS2 has also been shown to interact with ß-catenin [4]. A Rev-like leucine-rich, CRM1/exportin-regulated nuclear export signal (NES) sequence was identified within the carboxyl terminal region of LZTS2. Through this NES site, LZTS2 can modulate the export of nuclear ß-catenin, reducing the transcriptional activity of ß-catenin in the cell [4]. These data suggest that LZTS2 is a bona fide regulator of ß-catenin and plays critical role in development and tumorigenesis. The tumor suppressor PTEN is a phosphoprotein/phospholipid dual-specificity phosphatase [5]. Somatic mutation of PTEN frequently occurs in a variety of human tumors, including prostate cancer [6]. It has been shown that PTEN inhibits the activity of AKT/PKB, a key effector of the phosphatidylinositol 3-kinase (PI3K) signaling pathway, and functions as a tumor suppressor [7]. Activation of AKT can phosphorylate a number of downstream substrates, including glycogen synthase kinase 3ß, GSK3ß, [8,9]. Loss of PTEN increases GSK3ß phosphorylation and results in inhibiting ß-catenin degradation through the destruction complex [10].
Deletion of the human chromosomal 10q23-24 has been frequently observed in many human tumors, including prostate cancer. PTEN was identified within 10q23.3 region [11,12], and LZTS2 is located at 10q24.3, approximately 15Mb from the PTEN locus [1]. Intriguingly, both 10q23.3 and 10q24.3 regions, containing PTEN and LZTS2, are frequently deleted in a variety of human tumors [1,13]. PTEN deletion is closely associated with prostate cancer initiation and progression [6]. LZTS2 is expressed in human testis, prostate, and ovary tissues [4], and reduced expression of LZTS2 transcripts and proteins has been observed in prostate cancer tissues [3]. Similar to humans, in the mouse, Lzts2 is located on chromosome 19, only 11Mb from the mouse Pten gene [14]. In this study, we observed that PTEN and LZTS2 collaboratively enhance the transcriptional activity of ß-catenin in prostate cancer cells. To fully investigate the collaborative role of PTEN and LZTS2 in prostate tumor development, we generated a mouse model, in which both floxed Pten and Lzts2 alleles were targeted on chromosome 19. We subsequently crossed this mouse line with Probasin-Cre4 mice [15], and generated Lzts2 LoxP/LoxP :PB-Cre4, Pten loxP/Wt :PB-Cre4, and Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice. Using these mouse models, we characterized the biological consequences of the loss of either or both Pten and Lzts2 in the mouse prostatic luminal epithelium. We detected increased cellular proliferation in the prostates of Lzts2 LoxP/LoxP Pten loxP/Wt :PB-Cre4 compound mice, and observed accelerated tumor development and aggressive tumor invasion. These data elucidate a collaborative role of loss of both Pten and Lzts2 in prostate tumorigenesis, and implicate a critical role of Wnt/ß-catenin in prostate tumorigenesis.

Cell cultures and transfections
Human prostate cancer cell lines, PC3 and DU145, were maintained in DMEM supplemented with 5% fetal calf serum (FCS) (HyClone, Denver, CO). An AR-positive prostate cancer cell line, LNCaP, was maintained in T-medium (Invitrogen, Carlsbad, CA) with 5% FCS. Transient transfections were carried out using a LipofectAMINE transfection kit or LipofectA-MINE 2000 (Invitrogen, Carlsbad, CA). driven ß-galactosidase (ß-gal) reporter was generated by cloning the lacZ gene into the pcDNA3 vector [16]. The pcDNA-Tcf4 construct was provided by Dr. H. C. Clevers (CBG, Utrecht, The Netherlands). Expression constructs of human PTEN were generously provided by Dr. William Sellers (Dana-Farber Cancer Institute, Boston, MA). The full-length cDNA of human ß-catenin was cloned into pCDNA3 expression vector and mutants of ß-catenin with a single point mutation in the GSK3ß phosphorylation sites were generated by a PCR-based mutagenesis scheme as described previously [16]. LZTS2 expression vectors and shRNA pLentiviral vectors were generated as previously described [2,4].
Luciferase activity was measured in relative light units (RLU) as previously described [2,4,16,17]. Briefly, 50 μl of cell lysate was used for luciferase assays. The light output is measured after a 5 sec delay following injection of 50 μl luciferase buffer and 50 μl luciferin by the dual injector luminometer, according to the manufacturer's instructions (Analytical Luminesence Lab., San Diego, CA). The RLU from individual transfections were normalized by measurement of ß-galactosidase activity expressed from a co-transfected plasmid. Individual transfection experiments were done at least three times in triplicate and the results are reported as mean luciferase/ß -galactosidase (±SD) from representative experiments.
Protein fractions for immunoblotting were boiled in SDS-sample buffer and then resolved on a 10% SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane and probed with anti-ß-catenin antibody (Santa Cruz Biotechnology), anti-tubulin (clone DM1A, Neomarker), PCNA (PC10, Termo Fisher Scientific), or the polyclonal Lzts2 antibody [2]. Proteins were detected using the ECL kit (Amersham, Arlington Heights, IL). The antibody against tubulin (Neomarker, Fremont, CA) was used for protein loading.

Statistical analyses
We presented the data as the mean ±SD. We made comparisons between groups, using a twosided Student's t test. P<0.05 and P<0.01 were considered significant.

PTEN expression regulates ß-catenin transcriptional activity
It has been shown that wild-type PTEN expression inhibits the enhancement of ß-catenin mediated transcriptional activity in prostate cancer cells [10]. LZTS2 has also been shown to interact with ß-catenin and modulate the export of nuclear ß-catenin, reducing the transcriptional activity of ß-catenin [4]. In addition, both human and murine Pten and Lzts2 genes are closely localized on chromosome 10 or 19, respectively [14,24]. Furthermore, the deletion of both 10q23.3 and 10q24.3 regions that contain PTEN and LZTS2 genes have been frequently observed in a variety of human tumors [1,13]. Therefore, based on these lines of evidence, we examined the collaborative effect of PTEN and LZTS2 in regulating ß-catenin activity. We performed transient transfections in several prostate cancer cell-lines using either wild-type or stabilized mutant ß-catenin to assess PTEN expression in ß-catenin mediated transcription. These ß-catenin mutants contain point mutations within the phosphorylation site of GSK3β (S33F or S37A), which prevents degradation via the ubiquitin proteasome pathway. As shown in Fig 1, co-expression of TCF4 and ß-catenin induced transcription of the TOPflash (pGL3-OT) reporter in all three prostate cancer lines, including LNCaP, PC3, and DU145 (Fig 1A, 1B and 1C). Interestingly, a significant reduction of ß-catenin mediated transcriptional activity was observed when a wild-type PTEN vector was co-transfected with the wildtype ß-catenin expression vectors in all of three different cell lines (see lines 1 versus lines 2 in Fig 1A-1C, P<0.05). In contrast, there is almost no change in samples co-transfected with either stabilized mutant ß-catenin vectors in the presence or absence of PTEN (lines 3 to 6, Fig  1A-1C). The well described ß-catenin mutants used above are impervious to degradation by the destruction complex [25,26]. Therefore, these results suggest that PTEN can negatively regulate ß-catenin-mediated transcription in a GSK3ß-dependent manner.
PTEN and LZTS2 collaboratively regulate β-catenin transcriptional activity Next, we examined the possible collaborative effect of PTEN and LZTS2 on ß-catenin-mediated transcription. Co-expression of TCF4 and ß-catenin showed a transcriptional induction of pGL3-OT in LNCaP cells (Fig 2A). Transfection of PTEN or LZTS2 alone repressed wild type ß-catenin mediated transcriptional activity (lines 2 and 3, Fig 2A), while co-transfection of both PTEN and LZTS2 displayed significantly stronger repression (p<0.01, line 4 versus line 1, Fig 2A). In contrast, LZTS2 expression showed a repression on pGL3-OT promoter/ reporter mediated by both wild type and mutated ß-catenin (lines 7, 8, 11, and 12, Fig 2A). These data suggest that LZTS2 can repress ß-catenin mediated transcription collaboratively with PTEN, and its regulatory mechanism of ß-catenin is distinct from PTEN-mediated repression [4]. We then evaluated the repressive effect of endogenous LZTS2 using short hairpin RNA (shRNA) interference. Transfection of LZTS2 shRNA, but not control shRNA, showed reduced expression of endogenous LZTS2 proteins in LNCaP cells (Fig 2C). These knockdown effects also resulted in a dosage-dependent activation of both wild type and stabilized mutant ß-catenin with mutations of the serine residues on the pGL3-OT promoter/ reporter in LNCaP cells (Fig 2B). In contrast, there is no change in samples transfected with the control shRNA vector, suggesting that the above effect was due to LZTS2 knock-down. Taken together, these data demonstrate the role of LZTS2 in the regulation of ß-catenin-mediated transcription.

Generation of the Lzts2 and Pten compound mice
To further examine the collaborative role of PTEN and LZTS2 in vivo, we took a loss-offunction approach to directly address the biological significance of PTEN and LZTS2 in tumorigenesis using an Lzts2 and Pten deficient mouse strain. Because murine Lzts2 is located approximately 11Mb away from Pten [14], we recombined floxed Pten and Lzts2 loci into chromosome 19 by crossing Pten and Lzts2 floxed mice [2,19]. To examine the role of Pten and Lzts2 in the murine prostate, we subsequently crossed this mouse model with Probasin-Cre4 mice [15], and generated Lzts2 LoxP/LoxP :PB-Cre4, Pten loxP/Wt :PB-Cre4, and Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice (Fig 3A). Using specific primers (Fig 3A), we assessed mouse genotypes using genomic PCR analysis. We observed both appropriate floxed and deleted Pten and Lzts2 alleles in mouse prostate tissues (Fig 3B). We then evaluated Pten and Lzts2 expression in prostate tissues, which were isolated from 6-8 month old mice with different genotypes, using immunohistochemistry. As shown in Fig 3C, Lzts2 staining was observed in prostatic luminal cells of Pten loxP/Wt :PB-Cre4 mice, but very low or no staining with Lzts2 antibody was detected in samples isolated from Lzts2 LoxP/LoxP :PB-Cre4 and Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice. In a similar vein, decreased staining with a Pten  Conditional deletion of Lzts2 accelerates Pten-mediated oncogenic transformation in the mouse prostate Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 compound mice as well as Lzts2 LoxP/LoxP :PB-Cre4 and Pten loxP/Wt :PB-Cre4 mice were born at the expected Mendelian ratios and appeared normal with no obvious differences from their wild-type littermates at birth. We systematically examined male mice starting at 2-months of age and followed them until at least 16-months of age. We did not observe obvious abnormalities in 16 to 22-month-old Lzts2 LoxP/LoxP : PB-Cre4 mice (Fig 4A-4C'). Adhering to recommendations of the Mouse Models of Human Cancers Consortium Prostate Pathology Committee [20], we observed the development of prostatic intraepithelial neoplasia (PIN) in 6-month-old Pten loxP/Wt :PB-Cre4 mice. The PIN lesions first occurred in ventral prostate (VP), and then extended to dorsal (DP), lateral (LP), and anterior (AP) lobes. With time, these mPIN lesions progressed towards high-grade mPIN lesions or prostatic intracystic adenocarcinomas (Fig 4E-4F'). These lesions originated predominantly in the dorsal/lateral prostate (D/LP) and ventral prostate (VP) lobes, which is consistent with previous observations [19]. Notably, more Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 compound mice developed HGPIN lesions at 6-months of age than Pten loxP/Wt :PB-Cre4 mice (Fig 4G and 4G').

Identifying cellular origins of atypical and tumor cells
Mouse prostatic epithelium is composed of several cell types, including basal and luminal epithelial cells, as well as neuroendocrine cells. Previous studies have shown that luminal epithelial cell markers have been detected in PIN and prostatic adenocarcinoma lesions in Pten prostate conditional knockout mice with ARR2PB-Cre [19]. To determine the cellular origin of PIN in Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 compound mice, we performed comprehensive immunohistochemical analyses to examine a series of prostatic cellular markers on these high-grade PIN lesions (Fig 5). Atypical cells of PIN lesions failed to immunoreact with Lzts2 (Fig 5B1 and 5B3). Most atypical prostatic cells in the sample of Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice showed typical nuclear immunoreactivity with Ar (Fig 5C3), which is similar to the Pten loxP/Wt :PB-Cre4 mice (Fig 5C2). In samples isolated from both Pten loxP/Wt :PB-Cre4 and Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice, atypical cells showed positive immunoreactivity for E-cadherin and CK8, secretory epithelial markers (Fig 5D2, 5D3, 5E2 and 5E3), but showed no immunoreactivity for the neuroendocrine cell marker, synaptophysin (Fig 5H1-5H3). Immunoreactivity for CK5 and p63, the cellular markers for prostatic basal epithelial cells, appeared mainly in the basal compartment of normal prostatic glands, but rarely in atypical cells in the above mice (Fig 5F2, 5F3, 5G2 and 5G3). Taken together, these data demonstrate that prostatic atypical cells in Lzts2 LoxP/LoxP -Pten loxP/Wt : PB-Cre4 mainly contain luminal cellular markers.

Conditional deletion of Lzts2 enhances prostatic cell proliferation and results in alteration of ß-catenin subcellular localization
It has been shown that deletion of Pten enhances proliferation of prostatic epithelial cells in mice [19,27,28]. In this study, we assessed whether deletion of Lzts2 enhances cell proliferation in the prostate of mice using Ki67 immunohistochemistry. We carefully quantified Ki67 immunostaining in mouse prostate tissues by counting a total of 1000 epithelial cells from five high-power fields in samples isolated from Lzts2 LoxP/LoxP :PB-Cre4, Pten loxP/Wt :PB-Cre4, and Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice in different age groups. Experiments were repeated at least three times with three different slides prepared independently in each genotype. As shown in Fig 6A-6D, we presented data prepared from 6-8 month old mice with different genotypes mice. Heterozygous deletion of Pten appears to increases cell proliferation in prostatic epithelial cells in comparison with samples isolated from Lzts2 LoxP/LoxP :PB-Cre4 mice ( Fig  6B1 and 6B2 versus Fig 6A1 and 6A2). Intriguingly, a significant increase was observed in Ki67 immunostaining in both mPIN and prostatic adenocarcinoma lesions in Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 compound mice when compared to those in Pten loxP/Wt :PB-Cre4 mice (Fig 6C1 and 6C2 versus Fig 6B1 and 6B2). The epithelial proliferative index increased from 80 to 240 in HGPIN lesions (P<0.01, Fig 6D). These results demonstrate that Lzts2 deletion can augment the proliferation of prostatic epithelial cells mediated by Pten deletion in the compound mice. Previously, we have demonstrated that LZTS2 regulates the cellular level and localization of ß-catenin [4]. In this study, we also confirmed the effect of Lzts2 on cellular ß-catenin in mouse embryonic fibroblasts (MEFs). As shown in Fig 6E, both whole cell lysates and nuclear extracts prepared from different genotypes of MEFs were analyzed for levels of ß-catenin. A were prepared from different genotype embryos at E10.5. Either whole cell lysates or nuclear extracts were isolated from different genotype MEFs and analyzed by Western-blotting assays for either ß-catenin (ß-cat), tubulin, or PCNA. (F-H) Representative H&E and ß-catenin staining of Prostate tissues from the three different genotype mice is shown. Boxes highlight strong nuclear ß-catenin staining observed with conditional LZTS2 deletion (F2, H2). "*" or "**" means P<0.05 or <0.01, respectively.
https://doi.org/10.1371/journal.pone.0174357.g006 LZTS2 and PTEN regulate prostatic tumorigenesis notable increase of nuclear ß-catenin was observed in the nuclear extract of Lzts2 null MEFs despite similar levels of total ß-catenin in whole cell lysates isolated from the same cells. We then performed immunohistochemistry to assess ß-catenin expression in prostate tissues isolated from the three genotypes of mice. We observed typical cell membrane staining of ß-catenin in prostatic luminal cells in the samples isolated from all of three different genotype mice (Fig 6F2, 6G2, and 6H2)., Slightly increased cytoplasmic ß-catenin staining was observed in some of the prostatic epithelial cells of Pten loxP/Wt :PB-Cre4 mice (Fig 6G2). Intriguingly, a clear nuclear staining of ß-catenin appears in prostatic epithelial cells of samples from Lzts2 LoxP/LoxP : PB-Cre4 and Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice (Fig 6F2 and 6H2 boxed). These data further implicate the role of LZTS2 in promoting the nuclear export of ß-catenin in prostatic epithelial cells in mice.

Discussion
Human PTEN and LZTS2 are localized on the region of 10q23-24, within approximately 15Mb of each other [1]. Loss of heterozygosity (LOH) and homozygous deletions at human chromosomal region 10q23-24 are frequently found in prostate adenocarcinomas, as well as other malignancies, suggesting that multiple tumor suppressors may be present in the region [13]. Most intriguingly, approximately 10% of prostate tumor samples have been shown to possess both LZTS2 and PTEN deletion [29]. In this study, we generated a new mouse model in which both PTEN and LZTS2 were deleted simultaneously in prostatic epithelium to directly assess the biological significance and clinical relevance of PTEN and LZTS2 inactivation in prostate tumorigenesis. As we reported here, we observed accelerated oncogenic transformation and aggressive tumor phenotypes in the prostates of Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice with the deletion of both Pten and Lzts2 genes in comparison to Pten loxP/Wt :PB-Cre4 mice with Pten deletion only. Our data demonstrate the biological role of LZTS2 in tumorigenesis, and implicates the loss of both LZTS2 and PTEN as important biological and relevant events that can directly contribute to prostate cancer development and progression.
Interestingly, similar to humans, both murine Pten and Lzts2 are localized on Chromosome 19, only 11Mb apart from each other [14]. Homozygous deletion of Pten in the mouse embryo is lethal and characterized by developmental defects in the mesoderm, endoderm and ectoderm [30]. Heterozygous Pten mice develop multiple neoplasia in a wide spectrum of tissues including prostate, thyroid, colon, lymphatic system, mammary gland, and endometrium [30][31][32]. Conditional inactivation of Pten in the murine prostate results in PIN and invasive prostate cancer [19], suggesting a critical role between PTEN inactivation and prostate tumorigenesis. LZTS2 is expressed in testis, prostate, and ovary tissues [4], and reduced expression of LZTS2 transcripts and proteins has been observed in prostate cancer samples [3]. An increase in spontaneous tumor development has been observed in both aged Lzts2 heterozygous and homozygous knockout mice in comparison to wild type littermates [3]. These heterozygous or homozygous mice also showed an increase of BBN, a carcinogen, induced urinary bladder carcinoma development [3]. These lines of evidence suggest that both PTEN and LZTS2 play critical roles in tumorigenesis, and inactivation of both proteins may have a collaborative effect in oncogenic transformation. Our data presented in this report provide a line of evidence demonstrating combined loss of LZTS2 and PTEN as an important biological event in prostate cancer development and progression.
Multiple lines of evidence suggest that the Lzts2 gene is a tumor susceptibility gene [3]. Our previous data also showed a potential role of Lzts2 in prostate tumorigenesis. In this study, we also generated mice with conditional inactivation of Lzts2 in prostatic luminal epithelial cells using PB-Cre transgenic mice to directly examine Lzts2 in prostate tumorigenesis, [15]. We did not observe significant pathological changes in the prostate of both Lzts2 LoxP/wt :PB-Cre4 and Lzts2 LoxP/LoxP :PB-Cre4 mice up to 20-months of age (data not shown). These results imply that selective inactivation of Lzts2 in prostatic luminal epithelial cells by the ARR2PB promoter is insufficient to induce oncogenic transformation in prostatic luminal epithelial cells [15]. Homozygous deletion of Pten in the murine prostate results in invasive prostate cancer and metastatic prostate cancer of the lymph nodes and lung as early as ages of 2-months [19]. However, conditional heterozygous inactivation of Pten in the mouse prostate showed slow and moderate PIN and prostatic adenocarcinomas development [19]. Therefore, we used Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 compound mice to further evaluate the combined effect of Lzts2 and Pten inactivation in the prostate of mice. As detailed in this study, homozygous inactivation of Lzts2 in the mouse prostate accelerates the oncogenic transformation mediated by heterozygous loss of Pten in prostatic luminal epithelial cells. Given that PTEN loss of heterozygosity has been frequently observed in human tumors, Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice may mimic what occurs during the course of human prostate cancer development, and can be used to characterize this mechanism of prostate cancer initiation and progression. Specifically, identification of possible pathways and molecules that are involved in Lzts2 and Pten mediated tumorigenesis using the above mouse models would be biologically significant and clinical relevant.
Dysregulation of Wnt and ß-catenin mediated signaling pathways events in the pathogenesis of variety of human malignancies, including prostate cancer [33,34]. It has been shown that tumor cells contain high levels of nuclear ß-catenin through different regulatory mechanisms [35]. LZTS2 has been demonstrated to regulate ß-catenin nuclear export and modulate its cellular distribution and activity [4]. In this study, using Lzts2-deleted MEFs, we also assessed the effect of Lzts2 on the cellular localization of ß-catenin. Although we observed almost equal levels of ß-catenin in whole cell lysates prepared from either wild type or heterozygous and homozygous Lzts2 deletion MEFs, a significant increase of nuclear ß-catenin appears in Lzts2 null MEFs. This observation is consistent with previous data and demonstrates an important role of Lzts2 in regulating ß-catenin nuclear export [4]. PTEN exerts its function as a tumor suppressor through negative regulation of PI3K/AKT signaling pathways [5]. PI3K/Akt increases the stability of nuclear ß-catenin by phosphorylation and inactivation of the downstream substrate, GSK3ß, in prostate cancer cells, and PTEN deletion can augment PI3K/AKT action and increase cellular ß-catenin [10]. As shown in this study, prostate cancer cells co-transfected with both wild type PTEN and LZTS2 expression vectors showed less transcriptional activity of Tcf/ß-catenin than those transfected with either PTEN or LZTS2 alone. Interestingly, PTEN expression showed a much stronger inhibitory effect on wild type of ß-catenin than mutated ß-catenin. In contrast, LZTS2 expression inhibits both wild type and mutated ß-catenin activity. Through these distinct mechanisms, PTEN and LZTS2 collaboratively regulate cellular levels of ß-catenin and act as tumor suppressors to inhibit Wnt/ß-catenin-mediated oncogenic transformation in cells. In addition, we observed an increase in PIN and prostatic tumor development in Lzts2 LoxP/LoxP -Pten loxP/Wt : PB-Cre4 compound mice in comparison to Pten loxP/Wt :PB-Cre4 mice. Most atypical and tumor cells in Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice appear to be E-cadherin and CK8 positive, suggesting that they are of luminal epithelial cellular origin. In this study, we also measured cell proliferation in samples isolated from different mice. Prostatic luminal cells isolated from Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 compound mice appear more proliferative than those from other genotypes of mice. We also observed more cellular ß-catenin expression in atypical and tumor cells in the prostate of Pten loxP/Wt :PB-Cre4, and Lzts2 LoxP/LoxP -Pten loxP/Wt :PB-Cre4 mice. Interestingly, deletion of Lzts2 alone showed more nuclear ß-catenin expression than the other genotypes in the above samples. These data provide a link between increased cellular ß-catenin and oncogenic transformation in prostatic luminal epithelial cells. Validation of PTEN and LZTS2 loss, as well as cellular ß-catenin expression and localization within human tumor samples will provide useful information about the roles of PTEN and LZTS2 in human tumorigenesis; this knowledge may lead to the development of new therapeutic strategies for prostate cancer and other human malignancies.