Susan Kasper

Prostate Cancer Cells with Stem Cell Characteristics Reconstitute the Original Human Tumor In vivo

Cancer may arise from a cancer stem/progenitor cell that shares characteristics with its normal counterpart. We report the reconstitution of the original human prostate cancer specimen from epithelial cell lines (termed HPET for human prostate epithelial/hTERT) derived from this sample. These tumors can be described in terms of Gleason score, a classification not applied to any of the transgenic mouse models currently developed to mimic human disease. Immunohistochemical and Western blot analyses indicate that they do not express androgen receptor or p63, similar to that reported for prostate stem cells. These cell lines also express embryonic stem markers (Oct4, Nanog, and Sox2) as well as early progenitor cell markers (CD44 and Nestin) in vitro. Clonally derived HPET cells reconstitute the original human tumor in vivo and differentiate into the three prostate epithelial cell lineages, indicating that they arise from a common stem/progenitor cell. Serial transplantation experiments reconstitute the tumors, suggesting that a fraction of parental or clonally derived HPET cells have self-renewal potential. Thus, this model may enhance our understanding of human tumor development and provide a mechanism for studying cancer stem/progenitor cells in differentiation, tumorigenesis, preclinical testing, and the development of drug resistance.

15-Lipoxygenase-2 (15-LOX-2) Is Expressed in Benign Prostatic Epithelium and Reduced in Prostate Adenocarcinoma

Human 15S-lipoxygenase-2 (15-LOX-2) is a recently identified lipoxygenase that has approximately 40% sequence identity to the known human 5S-, 12S-, and 15S-lipoxygenases. 15-LOX-2 has a limited tissue distribution, with mRNA detected in prostate, lung, skin, and cornea, but not in numerous other tissues, including peripheral blood leukocytes. In the current study, we have characterized the distribution of 15-LOX-2 in the human prostate by immunohistochemistry, demonstrated the ability of benign prostate tissue to form 15S-hydroxyeicosatetraenoic acid (15S-HETE) from exogenous arachidonic acid (AA), and begun characterizing possible alterations in 15-LOX-2 in prostate adenocarcinoma. Incubation of benign prostate tissue with [14C]AA resulted in formation of [14C]15-HETE, as determined by reverse- and straight-phase high-performance liquid chromatography. 15-HETE was the major AA metabolite formed. By immunohistochemistry, 15-LOX-2 is located in secretory cells of peripheral zone glands and large prostatic ducts and somewhat less uniformly in apical cells of transition and central zone glands. 15-LOX-2 was not detected in the basal cell layer, stroma, ejaculatory ducts, seminal vesicles, or transitional epithelium. Immunostaining of 18 radical prostatectomy specimens showed a loss of 15-LOX-2 in the majority of prostate adenocarcinomas; 14 of 18 cases showed loss of 15-LOX-2 in >25% of the tumor (mean, 74.9% negative for 15-LOX-2; range, 38.9% to 100%). Incubation of paired pure benign and pure malignant prostate tissue from the same radical prostatectomies showed that 15-HETE formation was markedly reduced (>90%) or undetectable in incubations of prostate adenocarcinoma. 15-LOX-2 is a novel human lipoxygenase with a limited tissue distribution that is strongly expressed in benign prostate glandular epithelium and lost to a variable degree in the majority of prostate adenocarcinomas.

CD44 integrates signaling in normal stem cell, cancer stem cell and (pre)metastatic niches:

The stem cell niche provides a regulatory microenvironment for cells as diverse as totipotent embryonic stem cells to cancer stem cells (CSCs) which exhibit stem cell-like characteristics and have the capability of regenerating the bulk of tumor cells while maintaining self-renewal potential. The transmembrane glycoprotein CD44 is a common component of the stem cell niche and exists as a standard isoform (CD44s) and a range of variant isoforms (CD44v) generated though alternative splicing. CD44 modulates signal transduction through post-translational modifications as well as interactions with hyaluronan, extracellular matrix molecules and growth factors and their cognate receptor tyrosine kinases. While the function of CD44 in hematopoietic stem cells has been studied in considerable detail, our knowledge of CD44 function in tissue-derived stem cell niches remains limited. Here we review CD44s and CD44v in both hematopoietic and tissue-derived stem cell niches, focusing on their roles in regulating stem cell behavior including self-renewal and differentiation in addition to cell-matrix interactions and signal transduction during cell migration and tumor progression. Determining the role of CD44 and CD44v in normal stem cell, CSC and (pre)metastatic niches and elucidating their unique functions could provide tools and therapeutic strategies for treating diseases as diverse as fibrosis during injury repair to cancer progression.

NE-10 neuroendocrine cancer promotes the LNCaP xenograft growth in castrated mice.

Increases in neuroendocrine (NE) cells and their secretory products are closely correlated with tumor progression and androgen-independent prostate cancer. However, the mechanisms by which NE cells influence prostate cancer growth and progression, especially after androgen ablation therapy, are poorly understood. To investigate the role of NE cells on prostate cancer growth, LNCaP xenograft tumors were implanted into nude mice. After the LNCaP tumors were established, the NE mouse prostate allograft (NE-10) was implanted on the opposite flank of these nude mice to test whether NE tumor-derived systemic factors can influence LNCaP growth. Mice bearing LNCaP tumors with or without NE allografts were castrated 2 weeks after NE tumor inoculation, and changes in LNCaP tumor growth rate and gene expression were investigated. After castration, LNCaP tumor growth decreased in mice bearing LNCaP tumors alone, and this was accompanied by a loss of nuclear androgen receptor (AR) localization. In contrast, in castrated mice bearing both LNCaP and NE-10 tumors, LNCaP tumors continued to grow, had increased levels of nuclear AR, and secreted prostate-specific antigen. Therefore, in the absence of testicular androgens, NE secretions were sufficient to maintain LNCaP cell growth and androgen-regulated gene expression in vivo. Furthermore, in vitro experiments showed that NE secretions combined with low levels of androgens activated the AR, an effect that was blocked by the antiandrogen bicalutamide. Because an increase in AR level has been reported to be sufficient to account for hormone refractory prostate cancers, the NE cell population ability to increase AR level/activity can be another mechanism that allows prostate cancer to escape androgen ablation therapy.

Exploring the Origins of the Normal Prostate and Prostate Cancer Stem Cell

Prostate epithelial stem cells (PSCs) are primed by the urogenital mesenchyme to initiate bud formation and branching morphogenesis, ultimately culminating in a glandular structure composed of luminal, basal and neuroendocrine cells. Identity of this cell has remained elusive however cell populations enriched for cells exhibiting stem cell characteristics express the stem cell markers CD133+, α2β1hi, CD44 and Sca-1 along with embryonic stem cell factors including Oct-1, Nanog, Sox2 and nestin. Androgens are critical to prostate organogenesis and play a major role in normal prostate function and the development of prostate cancer. Cell lineage is another variable in the development of prostate cancer. This review discusses the embryonic prostate stem cell niche, normal prostate development, isolation and characterization of normal prostate and prostate cancer stem cells, and current concepts on the origin of prostate cancer.

Large fragment of the probasin promoter targets high levels of transgene expression to the prostate of transgenic mice

Androgen regulation and prostatic‐specific expression of targeted genes in transgenic mice can be controlled by a small DNA fragment of the probasin (PB) promoter (−426 +28 base pairs, bp). Although the small PB fragment was sufficient to direct prostate‐specific expression, the low levels of transgene expression suggested that important upstream regulatory sequences were missing.

The Loss of TGF-β Signaling Promotes Prostate Cancer Metastasis

In breast and colon cancers, transforming growth factor (TGF)-beta signaling initially has an antineoplastic effect, inhibiting tumor growth, but eventually exerts a proneoplastic effect, increasing motility and cancer spread. In prostate cancer, studies using human samples have correlated the loss of the TGF-beta type II receptor (T beta R II) with higher tumor grade. To determine the effect of an inhibited TGF-beta pathway on prostate cancer, we bred transgenic mice expressing the tumorigenic SV40 large T antigen in the prostate with transgenic mice expressing a dominant negative T beta R II mutant (DN II R) in the prostate. Transgene(s) and TGF-beta 1 expression were identified in the prostate and decreased protein levels of plasminogen activator inhibitor type I, as a marker for TGF-beta signaling, correlated with expression of the DN II R. Although the sizes of the neoplastic prostates were not enlarged, increased amounts of metastasis were observed in mice expressing both transgenes compared to age-matched control mice expressing only the large T antigen transgene. Our study demonstrates for the first time that a disruption of TGF-beta signaling in prostate cancer plays a causal role in promoting tumor metastasis.

DJ-1 Binds Androgen Receptor Directly and Mediates Its Activity in Hormonally Treated Prostate Cancer Cells

The oncogene DJ-1 has been associated with multiple cancers, including prostate cancer, where it can be stabilized by androgens and antiandrogens. However, little data exist on the expression pattern and function of DJ-1 in prostate cancer. To address the function of DJ-1 in prostate, a yeast two-hybrid screen was done to identify novel DJ-1 binding proteins. The androgen receptor (AR) was identified and confirmed as a DJ-1 binding partner. This is the first evidence that DJ-1 directly interacts with AR. We also show that modulation of DJ-1 expression regulated AR transcriptional activity. Importantly, both the subcellular localization of DJ-1 and the interaction with AR are regulated by androgens and antiandrogens. Additionally, immunohistochemical staining on two human prostate cancer tissue arrays was done providing the first large-scale expression analysis of DJ-1 in prostate. DJ-1 expression did not change with Gleason pattern but increased after androgen deprivation therapy, indicating that it may be involved in the development of androgen independence. These data provide a novel mechanism where DJ-1-mediated regulation of AR may promote the progression of prostate cancer to androgen independence.

Survey of genetically engineered mouse models for prostate cancer: analyzing the molecular basis of prostate cancer development, progression, and metastasis.

Genetically engineered mouse models have been generated to study the molecular basis of prostate cancer (PCa) development, progression, and metastasis. Selection of a prostate‐specific promoter, such as the probasin (PB) and prostate specific antigen (PSA) promoters, is critical for generating sufficient levels of transgene expression to elicit a phenotypic response. To date, target genes have included growth factors, cell cycle regulators, pro‐ and anti‐apoptotic proteins, steroid hormone and growth factor receptors, oncogenes, tumor suppressors, and homeobox genes. The experimental approaches used to generate these mouse models include overexpression of the transgene, knock‐out/knock‐in of transgene expression and conditional regulation of expression using Cre/lox technology. This review summarizes the promoters, which have been utilized to create genetically engineered mouse models for PCa. Furthermore, the effects of gene disruption on promoting low‐ and high‐grade intraepithelial neoplasia (LGPIN and HGPIN, respectively), locally invasive carcinoma and metastatic lesions will be discussed. To date, the PB‐Cre4 × PTENloxp/loxp model appears to be the only model that represents the entire continuum of prostate adenocarcinoma development, tumor progression, and metastasis, although models that develop prostatic neuroendocrine (NE) cancer can be generated by disrupting one genetic event. Indeed, analysis of bigenic mouse models indicates that two genetic events are generally required for progression from HGPIN to locally invasive adenocarcinoma and that two to five genetic events can promote metastasis to distant sites. Studying the effects of genetic perturbation on PCa biology will increase our understanding of the disease process and potentially provide targets for developing novel therapeutic approaches.

Monitoring Mouse Prostate Development by Profiling and Imaging Mass Spectrometry

Mass spectrometry-based tissue profiling and imaging are technologies that allow identification and visualization of protein signals directly on thin sections cut from fresh frozen tissue specimens. These technologies were utilized to evaluate protein expression profiles in the normal mouse prostate during development (1–5 weeks of age), at sexual maturation (6 weeks of age), and in adult prostate (at 10, 15, or 40 weeks of age). The evolution of protein expression during normal prostate development and maturation were subsequently compared with 15-week prostate tumors derived from genetically engineered mice carrying the Large T antigen gene under regulation of the prostate-specific probasin promoter (LPB-Tag mouse model for prostate cancer). This approach identified proteins differentially expressed at specific time points during prostate development. Furthermore expression of some of these proteins, for example probasin and spermine-binding protein, were associated with prostate maturation, and prostate tumor formation resulted in their loss of expression. Cyclophilin A, a protein found in other cancers, was differentially α-acetylated on the N terminus, and both isoforms appeared during normal prostate and prostate tumor development. Imaging mass spectrometry localized the protein signals to specific prostatic lobes or regions. Thus, tissue profiling and imaging can be utilized to analyze the ontogeny of protein expression during prostate morphogenesis and tumorigenesis and identify proteins that could potentially serve as biomarkers for prostate cancer.