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. Author manuscript; available in PMC: 2011 Jul 1.
Published in final edited form as: Cancer Biol Ther. 2010 Dec 1;10(11):1194–1200. doi: 10.4161/cbt.10.11.13814

Reduction of Pten dose leads to neoplastic development in multiple organs of PtenshRNA mice

Hong Shen-Li 1, Susan Koujak 1, Matthias Szablocs 2,4, Ramon Parsons 1,2,3,4,*
PMCID: PMC3018670  NIHMSID: NIHMS254407  PMID: 20980828

Abstract

To address the impact of partial reduction of Pten on tumor initiation, we generated PtenshRNA mice, in which PTEN expression was reduced below normal levels in various tissues. PtenshRNA mice frequently developed lymphoid and prostatic hyperplasia, splenomegaly and sebaceous adenomas. Our observations support the notion that partial reduction of the dose of Pten with shRNA is sufficient to induce neoplastic disease in multiple organ systems.

Keywords: pten, shRNA, RNAi, neoplasia, haploinsufficiency, tumor suppressor

Introduction

The tumor suppressor PTEN is one of the most commonly inactivated genes in human cancer. Germline PTEN mutations are associated with three human hamartoma syndromes (Cowden’s disease, Bannayan-Zonana syndrome and Lhermitte-Duclos disease) in which patients are predisposed to cancer. Somatic PTEN mutation/deletion is frequently detected in a wide variety of sporadic cancers.1–5 Biallelic inactivation of PTEN is an early event in uterine cancer but a late event in many advanced stage malignancies.3–6 In addition, partial inactivation of PTEN is an even more common occurrence and manifests itself in a variety of ways including deletion of one copy, reduced promoter function and inhibition of protein function. Extensive studies have suggested that PTEN exercises its tumor suppressor function mainly through negative regulation of the PDK1/PI3K/mTOR signaling pathway.7,8 The finding that either Pdk1 hypomorph or Akt1 deficiency is sufficient to suppress tumor development in Pten+/− mice has confirmed this notion and further points to Akt as one of the major down-stream effectors of Pten.9,10

Accumulating evidence supports the concept that loss of one of two copies of a tumor suppressor gene is an important mechanism for stimulating haplo-insufficient tumor progression.11–16 In such a model, the initial inactivation of one allele of a tumor suppressor gene confers on the mutant cells a selective growth advantage that leads to a neoplastic lesion. Upon this foundation, genetic or epigenetic lesions in other genes can accumulate to finally elicit a full-fledged tumor phenotype.11 Growing numbers of genes including p53,12 p27,13 Nkx3.1,14,15 and Dmp1,16 fall into this category.

Pten+/− mice develop neoplasia in multiple tissues including prostate, endometrium, adrenal medulla, lymph nodes, intestine, thyroid and mammary gland.17–19 Many of these tumors (lymph node, prostate, colon) retain one wild type allele and develop in a haplo-insufficient manner, while others (endometrium, adrenal medulla) undergo loss of the wild type allele and develop in a classical two-hit manner.17–27 In addition, analysis of a partially inactive hypomorphic Pten allele has shown that only a modest reduction of Pten dose is required to initiate tumors.28

To directly examine the hypothesis that Pten dosage affects tumor development, we utilized shRNA technology,29 which has the advantage that null tumors are extremely unlikely because both Pten alleles are intact. We generated multiple lines of PtenshRNA mice in which the level of Pten expression was reduced. As a result, these mice showed an increase in lymphoid and prostatic hyperplasia, brain size and sebaceous adenomas. These findings support the concept that partial reduction of Pten is sufficient to initiate tumor development.

Results

Generation of Pten altered mice using shRNA

We utilized a constitutively active human RNA polymerase III U6 promoter to express Pten-targeting shRNA in murine embryonic stem cells for the generation of transgenic mice, in which the expression of Pten would be reduced relative to wild type.30 Three shRNA expression pgk-neo resistance vectors (sh1, sh2 and sh3), targeting the sequences at the 5′ end, the 3′ end and the middle of the Pten coding region, respectively, were constructed. These vectors were introduced into the mouse ES cells and individual clones were screened for expression of PTEN protein. We were able to isolate multiple independent clones that had a reduction in Pten expression using the sh2 and sh3 hairpins but not the sh1 hairpin (Table 1).

Table 1.

Germline transmission of the shRNA-PTEN transgene in F1 progeny

ES clone Hairpin No. of F1 chimera Germline transmission Trangenic line
A sh2 2 Yes PtenshRNA4
B sh2 5 Yes PtenshRNA5
C sh3 4 Yes PtenshRNA21
D sh3 2 Sterile none
E sh3 1 Sterile none
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A summary of chimera production and subsequential germline transmission by selected shRNA-Pten ES clones. ES clones were screened for at least 50% inhibition of Pten expression level by western Blot.

Five ES clones derived from the sh2 (clones A and B) or sh3 (clones C, D and E) vector were injected into C57BL/6 blastocysts. As shown in Table 1, several high-percentage chimeras were obtained. Interestingly, male chimeras developed testicular masses that were confirmed to be testicular teratomas, a result that was previously observed in Pten+/− chimeras.17 This report is focused on lines derived from clone A (PtenshRNA4), clone B (PtenshRNA5) and clone C (PtenshRNA21) that had successful germline transmission of shRNA as assayed by PCR and Southern analysis.

Reduced PTEN expression and elevated Akt phosphorylation in PtenshRNA mouse tissues

Upon obtaining F1 animals, we examined the PTEN protein level in various organs. As shown in Figure 1, two transgenic PtenshRNA mouse lines (PtenshRNA4 and PtenshRNA5) generated from the sh2 hairpin showed marked reduction of PTEN expression in all organs examined including liver, heart, brain and uterus (Fig. 1A and B). With the exception of liver, phospho-AKT (serine 473) levels were upregulated when PTEN expression was reduced in the sh2 lines PtenshRNA4 and PtenshRNA5 (Fig. 1). One transgenic line (PtenshRNA21) was generated from the sh3 hairpin; it showed no reduction of Pten in the liver but reduction was seen in the other organs examined (Fig. 1C). Interestingly, for PtenshRNA21, phospho-AKT was not elevated as expected when Pten was reduced.

Figure 1.

Figure 1

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Pten expression level in tissues of PtenshRNA lines. Western blot was performed using total protein lysate of indicated organs from wildtype (WT) and PtenshRNA4 (4) (A), PtenshRNA5 (5) (B) and PtenshRNA21 (21) (C) to measure PTEN protein and phospho-473-AKT levels. Vinculin and total AKT protein were also tested as loading controls. Dashed lines indicate lanes that are juxtaposed for presentation from the same gel.

Reduction of Pten dosage causes decreased life span

We next examined the effect of altering the dose of Pten on mouse disease-free life span in our PtenshRNA transgenic lines. The transgenic mice were compared to wild type litter mates and Pten+/− mice. In order to further reduce the Pten level, we crossed PtenshRNA5 or PtenshRNA21 with Pten+/− and PtenshRNA lines with each other to generate PtenshRNA/shRNA offspring. During the course of the study, we noticed that the PtenshRNA5 and PtenshRNA4 lines did not follow the expected Mendelian ratio (1:1) when crossed with wild type (WT:PtenshRNA5 = 48:40; WT:PtenshRNA4 = 18:10). The offspring of the PtenshRNA4 and PtenshRNA5 cross also demonstrated a reduced viability of PtenshRNA carriers (WT:PtenshRNA5:PtenshRNA4: PtenshRNA4/5 = 28:14:16:5 vs. expected Mendelian ratio of 1:1:1:1). We generated survival plots for these lines as well as for wild type and Pten+/− mice. During the observation period of 50 weeks, both wild type (n = 59) as well as the PtenshRNA21 mice (n = 11) remained healthy. In contrast, PtenshRNA4 (n = 27), PtenshRNA5 (n = 35) lines, Pten+/− (n = 60), compound Pten+/−/shRNA (n = 9; n = 6) and compound PtenshRNA/shRNA (n = 20) mice all had reduced life-span (Fig. 2). It is interesting that although PtenshRNA4, PtenshRNA5 and PtenshRNA21 all had longer life spans than Pten+/− mice, both Pten+/−/shRNA5 and Pten+/−/shRNA21 mice had reduced life spans compared to Pten+/− alone. Also, mice containing double PtenshRNA (PtenshRNA5/21, n = 13; PtenshRNA4/5, n = 5; PtenshRNA4/21, n = 2) had shorter life spans than any line alone.

Figure 2.

Figure 2

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Survival analysis of PtenshRNA, Pten+/− and compound mutant mice. Mice from indicated lines (wildtype, WT; PtenshRNA4, 4; PtenshRNA5, 5; PtenshRNA21, 21; Pten+/−, HE T; Pten+/−/shRNA21, 21/HE T; Pten+/−/shRNA5, 5/HE T; Pten+/−/shRNA5/21, PtenshRNA4/5, PtenshRNA4/21, Double shRNA) were monitored until they reached end-points for sacrifice or one year of age. The sacrificed animals were subjected to gross pathological examination and histological analysis of all masses.

Reduction of Pten dosage results in neoplastic phenotypes that mimic phenotypes found in Pten+/− animals

We were most interested in understanding how Pten dosage affects cancer phenotypes in these mice. To address this question, a detailed histological and pathological survey has been made for all the single and double mutants between six months and one year of age. Significantly, all three lines of PtenshRNA mice showed evidence of lymphoid hyperplasia and splenomegaly (Table 2 and Fig. 3), similar to what was seen in Pten+/− mice.11–13 Enlarged hyperplastic lymph nodes were found in 50 out of 75 PtenshRNA animals analyzed. A similar frequency of splenomegaly was also observed. The pathological lesions were categorized as follicular hyperplasia (FH) and/or extramedullary hematopoeisis (EMH). Many mice died of the massive lymphadenopathy and splenomegaly, with PtenshRNA4 and PtenshRNA5 mice having a slightly higher incidence than observed in Pten+/−. In Pten+/−/shRNA5, Pten+/−/shRNA21 and PtenshRNA5/21 mice, an even greater incidence was observed (Fig. 3A). In addition, 24 out of 68 PtenshRNA mutant mice were found to have inflammation in the salivary gland (sialadenitis) (Table 2 and Fig. 3), which occurred in a Pten-dose dependent manner.

Table 2.

A summary of incidence of pathological phenotypes in a variety of PtenshRNA mouse organs

Tissues Pathology No. observed No. of PtenshRNA mice analyzed
Prostate Atypical hyperplasia (AH) and Prostatic intraepithelial neoplasia (PIN) 6 43
Uterus Complex atypical hyperplasia 1 33
Adrenal Pheochromocytoma 2 70
Lymph node Lymphoid hyperplasia 50 75
Spleen Splenomegaly with folicular hyperplasia (FH) or epithelial-mesenteric hyperplasia (EMH) 48 72
Salivary gland Sialadenitis (Inflammation of the salivary gland) 24 68
Skin Severe inflammatory ulcers, sebacious gland adenoma, sebacious cyst, dermoid cyst. 23 76
Brain 0 77
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A total of 77 mice of both sexes from PtenshRNA lines PtenshRNA4, PtenshRNA5 and PtenshRNA21 were analyzed histopathologically. Shown in the table are the total number of specimen analyzed and number with indicated phenotype.

Figure 3.

Figure 3

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Pathological phenotype in organs of individual mice lines. (A) Incidence of pathological alteration in organs of individual mice lines. The percentile in the Y-axis represents the proportion of animals in a certain mouse line with pathological phenotype in organs indicated in the X-axis. Each column represents an organ type of an individual mouse line, which was distinguished by fill-in colors as illustrated on the right side of the graph lines (wildtype, WT; PtenshRNA4, 4; PtenshRNA5, 5; PtenshRNA21, 21; Pten+/−, HE T; Pten+/−/shRNA21, 21/HE T; Pten+/−/shRNA5, 5/HE T; Pten+/−/shRNA5/21, 5/21). Numbers in the () indicates the total number of animals analyzed in each line. (B) Histopathology of phenotypes in skin (a and b), salivary gland (c), lymph node (d) and spleen (e) of PtenshRNA mice.

PtenshRNA mice had skin tumors that were not observed in Pten+/− mice. Interestingly, many PtenshRNA mice (23 out of 76) had severe skin lesions on the face, ear, neck or other parts of the body that were severely pruritic and required that the mice be sacrificed. Histological analysis of the lesions showed that they were associated with inflammatory cells and contained tumors of the hair follicle, sebaceous gland, sebaceous cysts and dermoid cysts (Fig. 3B). The origin of these tumors could be pluripotent dermal stem cells that retain the ability to form different dermal structures, but it is also possible that they are initiated by the inflammation. In some cases inflammation was severe and only ulcerative tissue could be detected. Interestingly, skin lesions were not observed in our Pten+/− controls, which suggests that dose reduction of Pten due to deletion of one allele is not sufficient to elicit this phenotype.

PtenshRNA mice show infrequent and mild change in the uterus and adrenal glands

As shown in Table 2, only one endometrial hyperplasia was found in 33 PtenshRNA mice. Also from a total of 70 animals, only 2 PtenshRNA4 mice had mild pheochromocytoma of the adrenal medulla. Even compound PtenshRNA5/21 mice had very low incidence of lesions in these organs (Fig. 3). In contrast, Pten+/− mice showed 100% for developing complex atypical endometrial hyperplasia of the uterus and 80% penetrance for pheochromocytoma (Fig. 3). Given that endometrial and adrenal tumors from Pten+/− mice undergo LOH of the wild type allele,11,12 these data suggest that inactivation of both Pten alleles are preferred for tumor initiation in these tissues but that partial reduction is sufficient although not highly penetrant.

PtenshRNA and prostate neoplasia

Unlike Pten+/− mice, which develop prostatic lesions ranging from hyperplasia to prostate carcinoma, a relatively small fraction of PtenshRNA mice developed prostatic lesions (Table 2 and Fig. 3A). As summarized in Table 2, prostate lesions were found in only 6 of the 43 shRNA mutants, 4 of which showed hyperplasia and 2 of which had early prostatic intraepithelial neoplasia (PIN) lesions. In contrast, Pten+/− mice (as well as Pten+/−shRNA double mutants) showed 100% penetrance for developing PIN. To better understand the discrepancy between the Pten+/− and PtenshRNA mice, we compared PTEN protein and phospho-AKT levels in the prostates of these mice. As shown in Figure 4A, prostates of all three PtenshRNA lines (4, 5 and 21) expressed less Pten than that of wildtype mice, yet the level of expression was significantly higher than that of Pten+/− mice. Phospho-AKT levels in prostates of shRNA mice were only slightly higher than wildtype but considerably lower than in Pten+/− mice (Fig. 4A). Immunohistochemical analysis of mouse PtenshRNA and Pten+/− prostates confirmed that the level of Pten expression in normal prostate epithelium was lower in PtenshRNA and Pten+/− samples than it was in wild type samples (Fig. 4B). Increased AKT phosphorylation in regions of PIN were seen in both Pten+/− and Pten+/−/shRNA21 mice. However, in PtenshRNA prostatic lesions, no increased Akt phosphorylation was observed by immunostaining (Fig. 4B). The modest phenotypic change in the prostates of PtenshRNA mice appears to be due to the correspondingly modest reduction of PTEN protein levels relative to those observed in Pten+/− prostates.

Figure 4.

Figure 4

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Pten expression and AKT Phosphorylation in prostates of PtenshRNA mice. (A) Western blot of prostate total protein lysates from wildtype (WT), Pten+/− (+/−) and PtenshRNA4 (4), PtenshRNA5 (5) and PtenshRNA21 (21) as indicated. (B) Immunohistochemistry analysis of Pten expression and AKT-473 phosphorylation in prostates of WT, Pten+/− and PtenshRNA mice.

Reduction of Pten dose in mice causes increased brain size

To assess the effect of Pten dosage on the brain, each brain was weighed prior to the pathological and biochemical assays. Consistent with the notion that Pten plays a critical role for normal brain development and size,31–33 the PtenshRNA mice as well as the compound mutant Pten+/−/shRNA21 mice had heavier brains than those of the wildtype littermates. Comparison of total brain protein showed that each of the shRNA lines had lower expression of PTEN relative to wild type and heterozygous mice (Fig. 5A). The double mutant Pten+/−/shRNA21 mice showed the largest brains, which were on average about 30% larger than the brains from wildtype mice (Fig. 5B). No tumors were observed (Table 2).

Figure 5.

Figure 5

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Pten expression and Akt Phosphorylation in brains of PtenshRNA mice. (A) Western blot analysis of brain total protein lysate from PtenshRNA lines as indicated. (B) Average brain weight in wildtype (WT), Pten+/− (+/−), PtenshRNA4 (4), PtenshRNA5 (5), PtenshRNA21 (21) and the compound mutant Pten+/−shRNA21 (±21) mice. The Y-axis represents brain weight while each column represents mouse genotype as indicated in the X-axis.

Discussion

The goal of this research is to assess unambiguously the role of Pten dose in neoplasia. To this end, we generated multiple PtenshRNA mouse lines, in which the level of Pten was reduced. The advantage of the shRNA system over classical genetic systems is that manipulation of the dose is achievable without the risk of somatic inactivation of both alleles of the gene through mechanisms such as loss of heterozygosity. PtenshRNA caused lesions of the spleen, lymph node, salivary gland, skin, prostate, uterus and adrenal gland. Examination of the phenotype spectrum in these mice indicated that the effect of PtenshRNA was variably penetrant in different tissues. Our positive findings from the PtenshRNA mice demonstrate that partial reduction of PTEN expression is indeed sufficient to induce the development of multiple types of neoplasia, particularly in the lymphoid system and skin. On the other hand, because we have seen that the U6 shRNA vectors affect the expression of PTEN in some organs and not others (Fig. 1), relatively weak tumor development could be a result of modest to no reduction of PTEN expression in those tissues. Data in the prostate, uterus and adrenal gland demonstrate that PtenshRNA is not as potent as germline heterozygous mutation of Pten in these organ systems. We suggest that this is a result of rather modest reduction of Pten expression in these organs, which we could document in the prostate and uterus. These results suggest that any mechanism that leads to a reduction of PTEN expression could stimulate the initiation of tumorigenesis in a susceptible cell and suggest that some tissues may favor “two-hit” inactivation of Pten for tumors to develop.

Materials and Methods

Short hairpin RNA (shRNA) vectors

Three shRNA expression vectors (sh1, sh2 and sh3) were constructed, targeting the sequences at the 5′ end, middle region and the 3′ end of the Pten coding region, respectively.29 For design and cloning of shRNA sequences, a PCR-based strategy was adopted from a protocol provided by Greg Hannon from Cold Spring Harbor Laboratories. For each shRNA, we designed a simple 29 bp hairpin with a 4 nt loop and used “PCR-Shag” to clone it into the neo-resistance vector pBSKS-Frt-Neo-Frt (gift from T. Ludwig, Columbia University). First, shRNA oligonucleotides were synthesized, onto which 21 bases of homology to the human U6 promoter were added. To link the U6 promoter to the hair-pins, a PCR reaction was performed using pGEM/U6 as the PCR SHAG template with the oligonucleotide HS9:5′-ATA AGA ATG CGG CCG CGA TTT AGG TGA CAC TAT AG-3′ as the forward primer and one of the hair-pin containing oligonucleotides as the reverse primers:

  • 5′-GGA ATT CAA AAA AGT CAA GTC TAA GCC GAA TCC ACC CCC TCG CAA GCT TCC AAG AGG ATG GAT TCG ACT TAG ACT TGA CTA GTA TAT GTG CTG CCG AAG C-3′ (sh1),

  • 5′-GGA ATT CAA AAA AGG TTC ATT CCC TGG ACC AGA GCC AGT GAT CAA GCT TCA CCA CTG ACT CTG ATC CAG AGA ATG AAC CTA GTA TAT GTG CTG CCG AAG C-3′ (sh2) and

  • 5′-GGA ATT CAA AAA AGG GCC CTG AAT TAG AAG AAT ACA TCT TCA CAA GCT TCT GAA GAT ATA TTC CTC CAA TTC AGG ACC CTA GTA TAT GTG CTG CCG AAG C-3′ (sh3).

The underlined are complimentary sequences and the Pten coding-strand homologue is bolded. PCR products were digested with Eco RI and Not I and cloned into pBSKS-Frt-Neo-Frt.

Generation of Pten hypomorphic mice by expressing Pten shRNA constitutively with the U6 promoter

The U6 hairpin vectors were introduced into the mouse ES cells by electroporation and individual stable clones were tested after selection with neomycin for the expression of the PTEN protein by western blot. Many ES clones showed reduced Pten expression. Several ES cell lines representing various Pten expression levels were chosen for injection into BL/6 blastocysts. For each clone, 5 cells were injected into each of 20–30 different blastocysts and transferred to 2–3 pseudo-pregnant females. Among the new born pups, several high-percentage chimeras were obtained. These chimeras were out-crossed to C57Bl6 and germline transmission of the shRNA-expression construct was detected by PCR and Southern analysis. Mice were crossed with our Pten+/− colony for comparison. 17 PtenshRNA mice could not be bred past the fourth generation. All mouse experiments were approved by the Institutional Animal Care and Use Committee.

Autopsy and histopathology

Animals were sacrificed and tissues were examined. Samples were fixed in 10% buffered formalin and embedded in paraffin. Sections (5 micrometer) were stained with hematoxylin and eosin (H&E) or with antibodies to PTEN (Cell signaling, 138G6) or phospho-473-AKT (Cell Signaling, 9271) according to standard protocols.

Protein analysis

For immunoblots, cells were lysed and collected in Laemmli sample buffer. Protein lysates (40 μg) were resolved by denaturing polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. Antibodies to phospho-473-Akt, Akt (Cell Signaling, 9271), PTEN (Cascade BioScience, 6H2.1), vinculin and tubulin (Covance) were used according to the manufacturer’s instructions. Blots were developed with horseradish peroxidase-conjugated secondary antibody using the enhanced chemiluminescence system (Amersham Pharmacia).

Acknowledgments

We thank Dr. Greg Hannon for providing advice and assistance for cloning shRNA vectors targeting Pten. This work is supported by NCI grant CA082783.

Abbreviations

PTEN

phosphatase and tensin homolog on chromosome 10

shRNA

short-hairpin ribonucleic acid

PI3K

phosphatidylinositol 3-kinase

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