The Pace of Prostatic Intraepithelial Neoplasia Development Is Determined by the Timing of Pten Tumor Suppressor Gene Excision

Loss of the PTEN tumor suppressor is a common occurrence in human prostate cancer, particularly in advanced disease. In keeping with its role as a pivotal upstream regulator of the phosphatidylinositol 3-kinase signaling pathway, experimentally-induced deletion of Pten in the murine prostate invariably results in neoplasia. However, and unlike humans where prostate tumorigenesis likely evolves over decades, disease progression in the constitutively Pten deficient mouse prostate is relatively rapid, culminating in invasive cancer within several weeks post-puberty. Given that the prostate undergoes rapid androgen-dependent growth at puberty, and that Pten excisions during this time might be especially tumorigenic, we hypothesized that delaying prostate-specific Pten deletions until immediately after puberty might alter the pace of tumorigenesis. To this end we generated mice with a tamoxifen-inducible Cre recombinase transgene enabling temporal control over prostate-specific gene alterations. This line was then interbred with mice carrying floxed Pten alleles. Despite evidence of increased Akt/mTOR/S6K axis activity at early time points in Pten-deficient epithelial cells, excisions induced in the post-pubertal (6 wk-old) prostate yielded gradual acquisition of a range of lesions. These progressed from pre-malignant changes (nuclear atypia, focal hyperplasia) and low grade prostatic intraepithelial neoplasia (PIN) at 16–20 wks post-tamoxifen exposure, to overtly malignant lesions by ∼1 yr of age, characterized by high-grade PIN and microinvasive carcinoma. In contrast, when Pten excisions were triggered in the pre-pubertal (2 week-old) prostate, neoplasia evolved over a more abbreviated time-frame, with a spectrum of premalignant lesions, as well as overt PIN and microinvasive carcinoma by 10–12 wks post-tamoxifen exposure. These results indicate that the developmental stage at which Pten deletions are induced dictates the pace of PIN development.     

Glutaredoxin 2 (Grx2) Gene Deletion Induces Early Onset of Age-dependent Cataracts in Mice

Glutaredoxin 2 (Grx2) is an isozyme of glutaredoxin1 (thioltransferase) present in the mitochondria and nucleus with disulfide reductase and peroxidase activities, and it controls thiol/disulfide balance in cells. In this study, we investigated whether Grx2 gene deletion could induce faster age-related cataract formation and elucidated the biochemical changes effected by Grx2 gene deletion that may contribute to lens opacity. Slit lamp was used to examine the lenses in Grx2 knock-out (KO) mice and age-matched wild-type (WT) mice ages 1 to 16 months. In the Grx2 null mice, the lens nuclear opacity began at 5 months, 3 months sooner than that of the control mice, and the progression of cataracts was also much faster than the age-matched controls. Lenses of KO mice contained lower levels of protein thiols and GSH with a significant accumulation of S-glutathionylated proteins. Actin, αA-crystallin, and βB2-crystallin were identified by Western blot and mass spectroscopy as the major S-glutathionylated proteins in the lenses of 16-month-old Grx2 KO mice. Compared with the WT control, the lens of Grx2 KO mice had only 50% of the activity in complex I and complex IV and less than 10% of the ATP pool. It was concluded that Grx2 gene deletion altered the function of lens structural proteins through S-glutathionylation and also caused severe disturbance in mitochondrial function. These combined alterations affected lens transparency.     

Erratum to: Systemic Gene Delivery Protects the Photoreceptors in the Retinal Degeneration Slow Mouse

Neurochemical Research -     

Accumulating Progenitor Cells in the Luminal Epithelial Cell Layer Are Candidate Tumor Initiating Cells in a Pten Knockout Mouse Prostate Cancer Model

The PSA-Cre;Pten-loxP/loxP mouse prostate cancer model displays clearly defined stages of hyperplasia and cancer. Here, the initial stages of hyperplasia development are studied. Immunohistochemical staining showed that accumulated pAkt⁺ hyperplastic cells overexpress luminal epithelial cell marker CK8, and progenitor cell markers CK19 and Sca-1, but not basal epithelial cell markers. By expression profiling we identified novel hyperplastic cell markers, including Tacstd2 and Clu. Further we showed that at young age prostates of targeted Pten knockout mice contained in the luminal epithelial cell layer single pAkt⁺ cells, which overexpressed CK8, Sca-1, Tacstd2 and Clu; basal epithelial cells were always pAkt⁻. Importantly, in the luminal epithelial cell layer of normal prostates we detected rare Clu⁺Tacstd2⁺Sca-1⁺ progenitor cells. These novel cells are candidate tumor initiating cells in Pten knockout mice. Remarkably, all luminal epithelial cells in the proximal region of normal prostates were Clu⁺Tacstd2⁺Sca-1⁺. However, in PSA-Cre;Pten-loxP/loxP mice, the proximal prostate does not contain hyperplastic foci. Small hyperplastic foci in prostates of PSA-Cre;Pten-loxP/+ mice found at old age, showed complete Pten inactivation and a progenitor marker profile. Finally, we present a novel model of prostate development and renewal, including lineage-specific luminal epithelial progenitor cells. It is proposed that Pten deficiency induces a shift in the balance of differentiation to proliferation in these cells.     

Germline Genetic Variation Modulates Tumor Progression and Metastasis in a Mouse Model of Neuroendocrine Prostate Carcinoma

Neuroendocrine (NE) differentiation has gained increased attention as a prostate cancer (PC) prognostic marker. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC. TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross recombinant inbred panel to address this. Tumor growth and metastasis burden were quantified in heterozygous transgene positive F1 male mice at 30 weeks of age. Compared to wild-type C57BL/6J-Tg(TRAMP)824Ng/J males, TRAMP x CAST/EiJ, TRAMP x NOD/ShiLtJ and TRAMP x NZO/HlLtJ F1 males displayed significant increases in tumor growth. Conversely, TRAMP x WSB/EiJ and TRAMP x PWK/PhJ F1 males displayed significant reductions in tumor growth. Interestingly, despite reduced tumor burden, TRAMP x WSB/EiJ males had an increased nodal metastasis burden. Patterns of distant pulmonary metastasis tended to follow the same patterns as that of local dissemination in each of the strains. All tumors and metastases displayed positive staining for NE markers, synaptophysin, and FOXA2. These experiments conclusively demonstrate that the introduction of germline variation by breeding modulates tumor growth, local metastasis burden, and distant metastasis frequency in this model of NEPC. These strains will be useful as model systems to facilitate the identification of germline modifier genes that promote the development of aggressive forms of PC.     

Conditional Ablation of the Choroideremia Gene Causes Age-Related Changes in Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm^Flox, Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch’s membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5–6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm^Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.     

Brca2 and Trp53 Deficiency Cooperate in the Progression of Mouse Prostate Tumourigenesis

Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches.     

A Novel Deletion Mutation in the Men1 Gene in a Patient with Prolactinoma and a Family History of Pancreatic Tumors

ObjectiveMultiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant tumor syndrome caused by mutations in the MEN1 gene. Mutations in this tumor suppressor gene are often associated with neuroendocrine tumors. Here we describe a novel deletion mutation at codon 304 in the MEN1 gene of a patient with a prolactinoma and strong family history of pancreatic tumors.MethodsWe describe the patient’s clinical course and mutational analysis and review the relevant literature. Results: A 30-year-old pregnant female was referred to our institution’s psychological department for treatment of depression. She had developed a prolactinoma at age 17 and was being treated with 1 mg/week of cabergoline. A medical interview revealed a family history of pancreatic islet cell and other tumors; her mother died of pancreatic cancer, her brother is living with gastrinoma, and her sister died of leiomyosarcoma. Extensive examinations performed after delivery, including laboratory tests and computed tomography (CT) scans, did not reveal any other tumors. Mutational analysis of the MEN1 gene identified a heterozygous deletion mutation (c911_914delAGGT) at codon 304. This mutation produces a frameshift at p.304Lys and might disturb the splicing of intron 6 due to the lack of a donor site. The predicted menin protein from the mutated allele is truncated at amino acid 328.ConclusionWe report a novel deletion mutation (c911_914delAGGT) in the MEN1 gene that was likely associated with the patient’s prolactinoma and her strong family history of pancreatic tumors. (Endocr Pract. 2014; 20:e162-e165)     

Mouse Fetal Liver Culture System to Dissect Target Gene Functions at the Early and Late Stages of Terminal Erythropoiesis

Erythropoiesis involves a dynamic process that begins with committed erythroid burst forming units (BFU-Es) followed by rapidly dividing erythroid colony forming units (CFU-Es). After CFU-Es, cells are morphologically recognizable and generally termed terminal erythroblasts. One of the challenges for the study of terminal erythropoiesis is the lack of experimental approaches to dissect gene functions in a chronological manner. In this protocol, we describe a unique strategy to determine gene functions in the early and late stages of terminal erythropoiesis. In this system, mouse fetal liver TER119 (mature erythroid cell marker) negative erythroblasts were purified and transduced with exogenous expression of cDNAs or small hairpin RNAs (shRNAs) for the genes of interest. The cells were subsequently cultured in medium containing growth factors other than erythropoietin (Epo) to maintain their progenitor stage for 12 hr while allowing the exogenous cDNAs or shRNAs to express. The cells were changed to Epo medium after 12 hr to induce cell differentiation and proliferation while the exogenous genetic materials were already expressed. This protocol facilitates analysis of gene functions in the early stage of terminal erythropoiesis. To study late stage terminal erythropoiesis, cells were immediately cultured in Epo medium after transduction. In this way, the cells were already differentiated to the late stage of terminal erythropoiesis when the transduced genetic materials were expressed. We recommend a general application of this strategy that would help understand detailed gene functions in different stages of terminal erythropoiesis.     

Rapamycin Inhibition of Polyposis and Progression to Dysplasia in a Mouse Model

Familial adenomatous polyposis (FAP) is often due to adenomatous polyposis coli (APC) gene germline mutations. Somatic APC defects are found in about 80% of colorectal cancers (CRCs) and adenomas. Rapamycin inhibits mammalian target of rapamycin (mTOR) protein, which is often expressed in human adenomas and CRCs. We sought to assess the effects of rapamycin in a mouse polyposis model in which both Apc alleles were conditionally inactivated in colon epithelium. Two days after inactivating Apc, mice were given rapamycin or vehicle in cycles of two weeks on and two weeks off. Polyps were scored endoscopically. Mice were euthanized at time points or when moribund, and tissue analyses were performed. In other studies, mice with demonstrable Apc-defective colon polyps were given rapamycin, followed by analysis of their colon tissues. The median survival of mice receiving rapamycin treatment cycles was 21.5 versus 6.5 weeks in control mice (p = 0.03), and rapamycin-treated mice had a significantly lower percentage of their colon covered with polyps (4.3+/− 2 vs 56.5+/− 10.8 percent, p = 0.001). Mice with Apc-deficient colon tissues that developed high grade dysplasia treated with rapamycin underwent treatment for significantly longer than mice treated with vehicle (15.8 vs 5.1 weeks, p = 0.003). In Apc-defective colon tissues, rapamycin treatment was linked to decreased levels of β-catenin and Sox9 at 7 weeks. Other effects of rapamycin in Apc-defectivecolon tissues included decreased proliferation and increased numbers of differentiated goblet cells at 7 weeks. Rapamycin did not affect β-catenin-regulated gene expression in cultured intestinal epithelial cells. Rapamycin has potent inhibitory effects in a mouse colon polyposis model, and mTOR inhibition is linked to decreased proliferation and increased expression of differentiation markers in Apc-mutant colon epithelium and delays development of dysplasia. Our findings highlight the possibility that mTOR inhibitors may have relevance for polyposis inhibition approaches in FAP patients.     

Data on the Recurrence of Breast Tumors Fit a Model in which Dormant Cells are Subject to Slow Attrition but Can Randomly Awaken to Become Malignant

We successfully modeled the recurrence of tumors in breast cancer patients, assuming that:(i) A breast cancer patient is likely to have some circulating metastatic cells, even after initial surgery. (ii) These metastatic cells are dormant. (iii) The dormant cells are subject to attrition by the body’s immune system, or by random apoptosis or senescence.(iv) Recurrence suppressor mechanisms exist. (v) When such genes are disabled by random mutations, the dormant metastatic cell is activated, and will develop to a cancer recurrence. The model was also fitted to data on the survival of pancreatic cancer patients. The time course of cancer recurrence in a group of poor prognosis breast cancer patients could not be linked to the over- (or under-) expression of any gene in the primary tumors from which the recurrent tumors derived. Thus, the recurrence of the tumor in breast cancer patients appears to be a random event. Inasmuch as the kinetics of cancer recurrence in published data sets closely follows the model found for the appearance of sporadic retinoblastoma, tumor recurrence could be triggered by mutations in awakening-suppressor mechanisms. The retinoblastoma tumor suppressor gene was identified by tracing its occurrence in familial retinoblastoma pedigrees. Will it be possible to track the postulated cancer recurrence, awakening suppressor gene(s) in early recurrence breast cancer patients?     

Loss of Survivin in the Prostate Epithelium Impedes Carcinogenesis in a Mouse Model of Prostate Adenocarcinoma

The inhibitor of apoptosis protein survivin is expressed in most cancers. Using the conditional PTEN deletion mouse model, we previously reported that survivin levels increase with prostate tumor growth. Here we evaluated the functional role of survivin in prostate tumor growth. First, we demonstrated that mice lacking the survivin gene in prostate epithelium were fertile and had normal prostate growth and development. We then serially, from about 10–56 weeks of age, evaluated histopathologic changes in the prostate of mice with PTEN deletion combined with survivin mono- or bi-allelic gene deletion. While within this time period most of the animals with wild-type or monoallelic survivin deletion developed adenocarcinomas, the most severe lesions in the biallelic survivin deleted mice were high-grade prostatic intra-epithelial neoplasia with distinct histopathology. Many atypical cells contained large hypertrophic cytoplasm and desmoplastic reaction in the prostatic intra-epithelial neoplasia lesions of this group was minimal until the late ages. A reduced proliferation index as well as apoptotic and senescent cells were detected in the lesions of mice with compound PTEN/survivin deficiency throughout the time points examined. Survivin deletion was also associated with reduced tumor expression of another inhibitor of apoptosis member, the X-linked inhibitor of apoptosis. Our findings suggest that survivin participates in the progression of prostatic intraepithelial neoplasia to adenocarcinoma, and that survivin interference at the prostatic intraepithelial neoplasia stages may be a potential therapeutic strategy to halt or delay further progression.     

Conditional Tek Promoter-Driven Deletion of Arginyltransferase in the Germ Line Causes Defects in Gametogenesis and Early Embryonic Lethality in Mice

Posttranslational protein arginylation mediated by Ate1 is essential for cardiovascular development, actin cytoskeleton functioning, and cell migration. Ate1 plays a role in the regulation of cytoskeleton and is essential for cardiovascular development and angiogenesis—capillary remodeling driven by in-tissue migration of endothelial cells. To address the role of Ate1 in cytoskeleton-dependent processes and endothelial cell function during development, we produced a conditional mouse knockout with Ate1 deletion driven by Tek endothelial receptor tyrosine kinase promoter expressed in the endothelium and in the germ line. Contrary to expectations, Tek-Ate1 mice were viable and had no visible angiogenesis-related phenotypes; however, these mice showed reproductive defects, with high rates of embryonic lethality in the second generation, at stages much earlier than the complete Ate1 knockout strain. While some of the early lethality originated from the subpopulation of embryos with homozygous Tek-Cre transgene—a problem that has not previously been reported for this commercial mouse strain—a distinct subpopulation of embryos had lethality at early post-implantation stages that could be explained only by a previously unknown defect in gametogenesis originating from Tek-driven Ate1 deletion in premeiotic germs cells. These results demonstrate a novel role of Ate1 in germ cell development.