Potential roles of FGF5 as a candidate therapeutic target in prostate cancer.

Fibroblast growth factor (FGF) is a secreted ligand that is widely expressed in embryonic tissues but its expression decreases with age. In the developing prostate, FGF5 has been proposed to interact with the Hedgehog (Hh) signaling pathway to guide mitogenic processes. In the adult prostate, the FGF/FGFR signaling axis has been implicated in prostate carcinogenesis, but focused studies on FGF5 functions in the prostate are limited. Functional studies completed in other cancer models point towards FGF5 overexpression as an oncogenic driver associated with stemness, metastatic potential, proliferative capacity, and increased tumor grade. In this review, we explore the significance of FGF5 as a therapeutic target in prostate cancer (PCa) and other malignancies; and we introduce a potential route of investigation to link FGF5 to benign prostatic hyperplasia (BPH). PCa and BPH are two primary contributors to the disease burden of the aging male population and have severe implications on quality of life, psychological wellbeing, and survival. The development of new FGF5 inhibitors could potentially alleviate the health burden of PCa and BPH in the aging male population.

Microglial responses to CSF1 overexpression do not promote the expansion of other glial lineages.

BACKGROUND: Colony-stimulating factor 1 (CSF1) expression in the central nervous system (CNS) increases in response to a variety of stimuli, and CSF1 is overexpressed in many CNS diseases. In young adult mice, we previously showed that CSF1 overexpression in the CNS caused the proliferation of IBA1+ microglia without promoting the expression of M2 polarization markers. METHODS: Immunohistochemical and molecular analyses were performed to further examine the impact of CSF1 overexpression on glia in both young and aged mice. RESULTS: As CSF1 overexpressing mice age, IBA1+ cell numbers are constrained by a decline in proliferation rate. Compared to controls, there were no differences in expression of the M2 markers ARG1 and MRC1 (CD206) in CSF1 overexpressing mice of any age, indicating that even prolonged exposure to increased CSF1 does not impact M2 polarization status in vivo. Moreover, RNA-sequencing confirmed the lack of increased expression of markers of M2 polarization in microglia exposed to CSF1 overexpression but did reveal changes in expression of other immune-related genes. Although treatment with inhibitors of the CSF1 receptor, CSF1R, has been shown to impact other glia, no increased expression of oligodendrocyte lineage or astrocyte markers was observed in CSF1 overexpressing mice. CONCLUSIONS: Our study indicates that microglia are the primary glial lineage impacted by CSF1 overexpression in the CNS and that microglia ultimately adapt to the presence of the CSF1 mitogenic signal.

Pexidartinib treatment in Alexander disease model mice reduces macrophage numbers and increases glial fibrillary acidic protein levels, yet has minimal impact on other disease phenotypes.

BACKGROUND: Alexander disease (AxD) is a rare neurodegenerative disorder that is caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), an intermediate filament that is primarily expressed by astrocytes. In AxD, mutant GFAP in combination with increased GFAP expression result in astrocyte dysfunction and the accumulation of Rosenthal fibers. A neuroinflammatory environment consisting primarily of macrophage lineage cells has been observed in AxD patients and mouse models. METHODS: To examine if macrophage lineage cells could serve as a therapeutic target in AxD, GFAP knock-in mutant AxD model mice were treated with a colony-stimulating factor 1 receptor (CSF1R) inhibitor, pexidartinib. The effects of pexidartinib treatment on disease phenotypes were assessed. RESULTS: In AxD model mice, pexidartinib administration depleted macrophages in the CNS and caused elevation of GFAP transcript and protein levels with minimal impacts on other phenotypes including body weight, stress response activation, chemokine/cytokine expression, and T cell infiltration. CONCLUSIONS: Together, these results highlight the complicated role that macrophages can play in neurological diseases and do not support the use of pexidartinib as a therapy for AxD.

CSF1 Overexpression Promotes High-Grade Glioma Formation without Impacting the Polarization Status of Glioma-Associated Microglia and Macrophages.

Current therapies for high-grade gliomas extend survival only modestly. The glioma microenvironment, including glioma-associated microglia/macrophages (GAM), is a potential therapeutic target. The microglia/macrophage cytokine CSF1 and its receptor CSF1R are overexpressed in human high-grade gliomas. To determine whether the other known CSF1R ligand IL34 is expressed in gliomas, we examined expression array data of human high-grade gliomas and performed RT-PCR on glioblastoma sphere-forming cell lines (GSC). Expression microarray analyses indicated that CSF1, but not IL34, is frequently overexpressed in human tumors. We found that while GSCs did express CSF1, most GSC lines did not express detectable levels of IL34 mRNA. We therefore studied the impact of modulating CSF1 levels on gliomagenesis in the context of the GFAP-V12Ha-ras-IRESLacZ (Ras*) model. Csf1 deficiency deterred glioma formation in the Ras* model, whereas CSF1 transgenic overexpression decreased the survival of Ras* mice and promoted the formation of high-grade gliomas. Conversely, CSF1 overexpression increased GAM density, but did not impact GAM polarization state. Regardless of CSF1 expression status, most GAMs were negative for the M2 polarization markers ARG1 and CD206; when present, ARG1(+) and CD206(+) cells were found in regions of peripheral immune cell invasion. Therefore, our findings indicate that CSF1 signaling is oncogenic during gliomagenesis through a mechanism distinct from modulating GAM polarization status. Cancer Res; 76(9); 2552-60. ©2016 AACR.

Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week.

During postnatal development, microglia, CNS resident innate immune cells, are essential for synaptic pruning, neuronal apoptosis and remodeling. During this period microglia undergo morphological and phenotypic transformations; however, little is known about how microglial number and density is regulated during postnatal CNS development. We found that after an initial increase during the first 14 postnatal days, microglial numbers in mouse brain began declining in the third postnatal week and were reduced by 50% by 6weeks of age; these "adult" levels were maintained until at least 9months of age. Microglial CD11b levels increased, whereas CD45 and ER-MP58 declined between P10 and adulthood, consistent with a maturing microglial phenotype. Our data indicate that both increased microglial apoptosis and a decreased proliferative capacity contribute to the developmental reduction in microglial numbers. We found no correlation between developmental reductions in microglial numbers and brain mRNA levels of Cd200, Cx3Cl1, M-Csf or Il-34. We tested the ability of M-Csf-overexpression, a key growth factor promoting microglial proliferation and survival, to prevent microglial loss in the third postnatal week. Mice overexpressing M-Csf in astrocytes had higher numbers of microglia at all ages tested. However, the developmental decline in microglial numbers still occurred, suggesting that chronically elevated M-CSF is unable to overcome the developmental decrease in microglial numbers. Whereas the identity of the factor(s) regulating microglial number and density during development remains to be determined, it is likely that microglia respond to a "maturation" signal since the reduction in microglial numbers coincides with CNS maturation.

Sleeping Beauty mouse models identify candidate genes involved in gliomagenesis.

Genomic studies of human high-grade gliomas have discovered known and candidate tumor drivers. Studies in both cell culture and mouse models have complemented these approaches and have identified additional genes and processes important for gliomagenesis. Previously, we found that mobilization of Sleeping Beauty transposons in mice ubiquitously throughout the body from the Rosa26 locus led to gliomagenesis with low penetrance. Here we report the characterization of mice in which transposons are mobilized in the Glial Fibrillary Acidic Protein (GFAP) compartment. Glioma formation in these mice did not occur on an otherwise wild-type genetic background, but rare gliomas were observed when mobilization occurred in a p19Arf heterozygous background. Through cloning insertions from additional gliomas generated by transposon mobilization in the Rosa26 compartment, several candidate glioma genes were identified. Comparisons to genetic, epigenetic and mRNA expression data from human gliomas implicates several of these genes as tumor suppressor genes and oncogenes in human glioblastoma.

CSF1 overexpression has pleiotropic effects on microglia in vivo.

Macrophage colony stimulating factor (CSF1) is a cytokine that is upregulated in several diseases of the central nervous system (CNS). To examine the effects of CSF1 overexpression on microglia, transgenic mice that overexpress CSF1 in the glial fibrillary acidic protein (GFAP) compartment were generated. CSF1 overexpressing mice have increased microglial proliferation and increased microglial numbers compared with controls. Treatment with PLX3397, a small molecule inhibitor of the CSF1 receptor CSF1R and related kinases, decreases microglial numbers by promoting microglial apoptosis in both CSF1 overexpressing and control mice. Microglia in CSF1 overexpressing mice exhibit gene expression profiles indicating that they are not basally M1 or M2 polarized, but they do have defects in inducing expression of certain genes in response to the inflammatory stimulus lipopolysaccharide. These results indicate that the CSF1 overexpression observed in CNS pathologies likely has pleiotropic influences on microglia. Furthermore, small molecule inhibition of CSF1R has the potential to reverse CSF1-driven microglial accumulation that is frequently observed in CNS pathologies, but can also promote apoptosis of normal microglia.

Rosa26-GFP direct repeat (RaDR-GFP) mice reveal tissue- and age-dependence of homologous recombination in mammals in vivo.

Homologous recombination (HR) is critical for the repair of double strand breaks and broken replication forks. Although HR is mostly error free, inherent or environmental conditions that either suppress or induce HR cause genomic instability. Despite its importance in carcinogenesis, due to limitations in our ability to detect HR in vivo, little is known about HR in mammalian tissues. Here, we describe a mouse model in which a direct repeat HR substrate is targeted to the ubiquitously expressed Rosa26 locus. In the Rosa26 Direct Repeat-GFP (RaDR-GFP) mice, HR between two truncated EGFP expression cassettes can yield a fluorescent signal. In-house image analysis software provides a rapid method for quantifying recombination events within intact tissues, and the frequency of recombinant cells can be evaluated by flow cytometry. A comparison among 11 tissues shows that the frequency of recombinant cells varies by more than two orders of magnitude among tissues, wherein HR in the brain is the lowest. Additionally, de novo recombination events accumulate with age in the colon, showing that this mouse model can be used to study the impact of chronic exposures on genomic stability. Exposure to N-methyl-N-nitrosourea, an alkylating agent similar to the cancer chemotherapeutic temozolomide, shows that the colon, liver and pancreas are susceptible to DNA damage-induced HR. Finally, histological analysis of the underlying cell types reveals that pancreatic acinar cells and liver hepatocytes undergo HR and also that HR can be specifically detected in colonic somatic stem cells. Taken together, the RaDR-GFP mouse model provides new understanding of how tissue and age impact susceptibility to HR, and enables future studies of genetic, environmental and physiological factors that modulate HR in mammals.

A dominantly acting murine allele of Mcm4 causes chromosomal abnormalities and promotes tumorigenesis.

Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. Sdl mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from Sdl mice harbor small amplifications and deletions, including activating deletions at the Notch1 locus. Using exome sequencing it was determined that Sdl mice harbor a spontaneously acquired mutation in Mcm4 (Mcm4(D573H)). MCM4 is part of the heterohexameric complex of MCM2-7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of Mcms, and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias. Studies in Saccharomyces cerevisiae indicate that the Sdl mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in Sdl mice result from the ability of MCM4(D573H) to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities.

The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.

An insertional mutagenesis screen identifies genes that cooperate with Mll-AF9 in a murine leukemogenesis model.

Patients with a t(9;11) translocation (MLL-AF9) develop acute myeloid leukemia (AML), and while in mice the expression of this fusion oncogene also results in the development of myeloid leukemia, it is with long latency. To identify mutations that cooperate with Mll-AF9, we infected neonatal wild-type (WT) or Mll-AF9 mice with a murine leukemia virus (MuLV). MuLV-infected Mll-AF9 mice succumbed to disease significantly faster than controls presenting predominantly with myeloid leukemia while infected WT animals developed predominantly lymphoid leukemia. We identified 88 candidate cancer genes near common sites of proviral insertion. Analysis of transcript levels revealed significantly elevated expression of Mn1, and a trend toward increased expression of Bcl11a and Fosb in Mll-AF9 murine leukemia samples with proviral insertions proximal to these genes. Accordingly, FOSB and BCL11A were also overexpressed in human AML harboring MLL gene translocations. FOSB was revealed to be essential for growth in mouse and human myeloid leukemia cells using shRNA lentiviral vectors in vitro. Importantly, MN1 cooperated with Mll-AF9 in leukemogenesis in an in vivo BM viral transduction and transplantation assay. Together, our data identified genes that define transcription factor networks and important genetic pathways acting during progression of leukemia induced by MLL fusion oncogenes.

Clonal selection drives genetic divergence of metastatic medulloblastoma.

Medulloblastoma, the most common malignant paediatric brain tumour, arises in the cerebellum and disseminates through the cerebrospinal fluid in the leptomeningeal space to coat the brain and spinal cord. Dissemination, a marker of poor prognosis, is found in up to 40% of children at diagnosis and in most children at the time of recurrence. Affected children therefore are treated with radiation to the entire developing brain and spinal cord, followed by high-dose chemotherapy, with the ensuing deleterious effects on the developing nervous system. The mechanisms of dissemination through the cerebrospinal fluid are poorly studied, and medulloblastoma metastases have been assumed to be biologically similar to the primary tumour. Here we show that in both mouse and human medulloblastoma, the metastases from an individual are extremely similar to each other but are divergent from the matched primary tumour. Clonal genetic events in the metastases can be demonstrated in a restricted subclone of the primary tumour, suggesting that only rare cells within the primary tumour have the ability to metastasize. Failure to account for the bicompartmental nature of metastatic medulloblastoma could be a major barrier to the development of effective targeted therapies.

Sleeping beauty-mediated somatic mutagenesis implicates CSF1 in the formation of high-grade astrocytomas.

The Sleeping Beauty (SB) transposon system has been used as an insertional mutagenesis tool to identify novel cancer genes. To identify glioma-associated genes, we evaluated tumor formation in the brain tissue from 117 transgenic mice that had undergone constitutive SB-mediated transposition. Upon analysis, 21 samples (18%) contained neoplastic tissue with features of high-grade astrocytomas. These tumors expressed glial markers and were histologically similar to human glioma. Genomic DNA from SB-induced astrocytoma tissue was extracted and transposon insertion sites were identified. Insertions in the growth factor gene Csf1 were found in 13 of the 21 tumors (62%), clustered in introns 5 and 8. Using reverse transcription-PCR, we documented increased Csf1 RNAs in tumor versus adjacent normal tissue, with the identification of transposon-terminated Csf1 mRNAs in astrocytomas with SB insertions in intron 8. Analysis of human glioblastomas revealed increased levels of Csf1 RNA and protein. Together, these results indicate that SB-insertional mutagenesis can identify high-grade astrocytoma-associated genes and they imply an important role for CSF1 in the development of these tumors.

Novel candidate cancer genes identified by a large-scale cross-species comparative oncogenomics approach.

Comparative genomic hybridization (CGH) can reveal important disease genes but the large regions identified could sometimes contain hundreds of genes. Here we combine high-resolution CGH analysis of 598 human cancer cell lines with insertion sites isolated from 1,005 mouse tumors induced with the murine leukemia virus (MuLV). This cross-species oncogenomic analysis revealed candidate tumor suppressor genes and oncogenes mutated in both human and mouse tumors, making them strong candidates for novel cancer genes. A significant number of these genes contained binding sites for the stem cell transcription factors Oct4 and Nanog. Notably, mice carrying tumors with insertions in or near stem cell module genes, which are thought to participate in cell self-renewal, died significantly faster than mice without these insertions. A comparison of the profile we identified to that induced with the Sleeping Beauty (SB) transposon system revealed significant differences in the profile of recurrently mutated genes. Collectively, this work provides a rich catalogue of new candidate cancer genes for functional analysis.

Whole-body sleeping beauty mutagenesis can cause penetrant leukemia/lymphoma and rare high-grade glioma without associated embryonic lethality.

The Sleeping Beauty (SB) transposon system has been used as a somatic mutagen to identify candidate cancer genes. In previous studies, efficient leukemia/lymphoma formation on an otherwise wild-type genetic background occurred in mice undergoing whole-body mobilization of transposons, but was accompanied by high levels of embryonic lethality. To explore the utility of SB for large-scale cancer gene discovery projects, we have generated mice that carry combinations of different transposon and transposase transgenes. We have identified a transposon/transposase combination that promotes highly penetrant leukemia/lymphoma formation on an otherwise wild-type genetic background, yet does not cause embryonic lethality. Infiltrating gliomas also occurred at lower penetrance in these mice. SB-induced or accelerated tumors do not harbor large numbers of chromosomal amplifications or deletions, indicating that transposon mobilization likely promotes tumor formation by insertional mutagenesis of cancer genes, and not by promoting wide-scale genomic instability. Cloning of transposon insertions from lymphomas/leukemias identified common insertion sites at known and candidate novel cancer genes. These data indicate that a high mutagenesis rate can be achieved using SB without high levels of embryonic lethality or genomic instability. Furthermore, the SB system could be used to identify new genes involved in lymphomagenesis/leukemogenesis.

Identification of PDE4D as a proliferation promoting factor in prostate cancer using a Sleeping Beauty transposon-based somatic mutagenesis screen.

Retroviral and transposon-based mutagenesis screens in mice have been useful for identifying candidate cancer genes for some tumor types. However, many of the organs that exhibit the highest cancer rates in humans, including the prostate, have not previously been amenable to these approaches. This study shows for the first time that the Sleeping Beauty transposon system can be used to identify candidate prostate cancer genes in mice. Somatic mobilization of a mutagenic transposon resulted in focal epithelial proliferation and hyperplasia in the prostate. Efficient methods were established to identify transposon insertion sites in these lesions, and analysis of transposon insertions identified candidate prostate cancer genes at common insertion sites, including Pde4d. PDE4D was also overexpressed in human prostate cancer patient samples and cell lines, and changes in PDE4D mRNA isoform expression were observed in human prostate cancers. Furthermore, knockdown of PDE4D reduced the growth and migration of prostate cancer cells in vitro, and knockdown of PDE4D reduced the growth and proliferation rate of prostate cancer xenografts in vivo. These data indicate that PDE4D functions as a proliferation promoting factor in prostate cancer, and the Sleeping Beauty transposon system is a useful tool for identifying candidate prostate cancer genes.

A transposon-based genetic screen in mice identifies genes altered in colorectal cancer.

Human colorectal cancers (CRCs) display a large number of genetic and epigenetic alterations, some of which are causally involved in tumorigenesis (drivers) and others that have little functional impact (passengers). To help distinguish between these two classes of alterations, we used a transposon-based genetic screen in mice to identify candidate genes for CRC. Mice harboring mutagenic Sleeping Beauty (SB) transposons were crossed with mice expressing SB transposase in gastrointestinal tract epithelium. Most of the offspring developed intestinal lesions, including intraepithelial neoplasia, adenomas, and adenocarcinomas. Analysis of over 16,000 transposon insertions identified 77 candidate CRC genes, 60 of which are mutated and/or dysregulated in human CRC and thus are most likely to drive tumorigenesis. These genes include APC, PTEN, and SMAD4. The screen also identified 17 candidate genes that had not previously been implicated in CRC, including POLI, PTPRK, and RSPO2.

A conditional transposon-based insertional mutagenesis screen for genes associated with mouse hepatocellular carcinoma.

We describe a system that permits conditional mobilization of a Sleeping Beauty (SB) transposase allele by Cre recombinase to induce cancer specifically in a tissue of interest. To demonstrate its potential for developing tissue-specific models of cancer in mice, we limit SB transposition to the liver by placing Cre expression under the control of an albumin enhancer/promoter sequence and screen for hepatocellular carcinoma (HCC)-associated genes. From 8,060 nonredundant insertions cloned from 68 tumor nodules and comparative analysis with data from human HCC samples, we identify 19 loci strongly implicated in causing HCC. These encode genes, such as EGFR and MET, previously associated with HCC and others, such as UBE2H, that are potential new targets for treating this neoplasm. Our system, which could be modified to drive transposon-based insertional mutagenesis wherever tissue-specific Cre expression is possible, promises to enhance understanding of cancer genomes and identify new targets for therapeutic development.

Transposon-mediated mutagenesis in somatic cells: identification of transposon-genomic DNA junctions.

Understanding the genetic basis for tumor formation is crucial for treating cancer. Forward genetic screens using insertional mutagenesis technologies have identified many important tumor suppressor genes and oncogenes in mouse models of human cancer. Traditionally, retroviruses have been used for this purpose, allowing the identification of genes that can cause various forms of leukemia or lymphoma with murine leukemia viruses or mammary cancer with mouse mammary tumor viruses. Recently, the Sleeping Beauty transposon system has emerged as a tool for cancer gene discovery in mouse models of human cancer. Transposons mobilized in the mouse soma can insertionally mutate cancer genes, and the transposon itself serves as a molecular "tag," which facilitates candidate cancer gene identification. We provide an overview of some general issues related to use of Sleeping Beauty for cancer genetic studies and present here the polymerase chain reaction-based method for cloning transposon-tagged sequences from tumors.

Transposons for cancer gene discovery: Sleeping Beauty and beyond.

The use of Sleeping Beauty transposons as somatic mutagens to discover cancer genes in hematopoietic tumors and sarcomas has been documented. Here, we discuss the future of Sleeping Beauty for cancer genetic studies and the potential use of additional transposable elements for somatic mutagenesis.