Concurrent Olaparib and Radiation Therapy in Older Patients With Newly Diagnosed Glioblastoma: The Phase 1 Dose-Escalation PARADIGM Trial.

PURPOSE: Patients with glioblastoma who are older or have poor performance status (PS) experience particularly poor clinical outcomes. At the time of study initiation, these patients were treated with short-course radiation therapy (40 Gy in 15 fractions). Olaparib is an oral inhibitor of the DNA repair enzyme poly (ADP-ribose) polymerase (PARP) that is well tolerated as a single agent but exacerbates acute radiation toxicity in extracranial sites. Preclinical data predicted that PARP inhibitors would enhance radiosensitivity in glioblastoma without exacerbating adverse effects on the normal brain. METHODS AND MATERIALS: Phase 1 of the PARADIGM trial was a 3+3 dose-escalation study testing olaparib in combination with radiation therapy (40 Gy 15 fractions) in patients with newly diagnosed glioblastoma who were unsuitable for radical treatment either because they were aged 70 years or older (World Health Organization PS 0-1) or aged 18 to 69 years with PS 2. The primary outcome was the recommended phase 2 dose of olaparib. Secondary endpoints included safety and tolerability, overall survival, and progression-free survival. Effects on cognitive function were assessed via the Mini Mental State Examination. RESULTS: Of 16 eligible patients (56.25% male; median age, 71.5 years [range, 44-78]; 75% PS 0-1), 1 dose-limiting toxicity was reported (grade 3 agitation). Maximum tolerated dose was not reached and the recommended phase 2 dose was determined as 200 mg twice daily. Median overall survival and progression-free survival were 10.8 months (80% CI, 7.3-11.4) and 5.5 months (80% CI, 3.9-5.9), respectively. Mini Mental State Examination plots indicated that cognitive function was not adversely affected by the olaparib-radiation therapy combination. CONCLUSIONS: Olaparib can be safely combined with hypofractionated brain radiation therapy and is well tolerated in patients unsuitable for radical chemoradiation. These results enabled initiation of a randomized phase 2 study and support future trials of PARP inhibitors in combination with radiation therapy for patients with brain tumors.

Low-Dose Lung Radiation Therapy for COVID-19 Lung Disease: A Preclinical Efficacy Study in a Bleomycin Model of Pneumonitis.

PURPOSE: Low-dose whole lung radiation therapy (LDLR) has been proposed as a treatment for patients with acute respiratory distress syndrome associated with SARS-CoV-2 infection, and clinical trials are underway. There is an urgent need for preclinical evidence to justify this approach and inform dose, scheduling, and mechanisms of action. METHODS AND MATERIALS: Female C57BL/6 mice were treated with intranasal bleomycin sulfate (7.5 or 11.25 units/kg, day 0) and then exposed to whole lung radiation therapy (0.5, 1.0, or 1.5 Gy, or sham; day 3). Bodyweight was measured daily, and lung tissue was harvested for histology and flow cytometry on day 10. Computed tomography lung imaging was performed before radiation (day 3) and pre-endpoint (day 10). RESULTS: Bleomycin caused pneumonitis of variable severity, which correlated with weight loss. LDLR at 1.0 Gy was associated with a significant increase in the proportion of mice recovering to 98% of initial bodyweight, and a proportion of these mice exhibited less severe histopathologic lung changes. Mice experiencing moderate initial weight loss were more likely to respond to LDLR than those experiencing severe initial weight loss. In addition, LDLR (1.0 Gy) significantly reduced bleomycin-induced increases in interstitial macrophages, CD103+ dendritic cells (DCs), and neutrophil-DC hybrids. Overall, bleomycin-treated mice exhibited significantly higher percentages of nonaerated lung in left than right lungs, and LDLR (1.0 Gy) limited further reductions in aerated lung volume in right but not left lungs. LDLR at 0.5 and 1.5 Gy did not improve bodyweight, flow cytometric, or radiologic readouts of bleomycin-induced pneumonitis. CONCLUSIONS: Our data support the concept that LDLR can ameliorate acute inflammatory lung injury, identify 1.0 Gy as the most effective dose, and provide evidence that it is more effective in the context of moderate than severe pneumonitis. Mechanistically, LDLR at 1.0 Gy significantly suppressed bleomycin-induced accumulation of pulmonary interstitial macrophages, CD103+ DCs, and neutrophil-DC hybrids.

Pharmacokinetics, safety, and tolerability of olaparib and temozolomide for recurrent glioblastoma: results of the phase I OPARATIC trial.

BACKGROUND: The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib potentiated radiation and temozolomide (TMZ) chemotherapy in preclinical glioblastoma models but brain penetration was poor. Clinically, PARP inhibitors exacerbate the hematological side effects of TMZ. The OPARATIC trial was conducted to measure penetration of recurrent glioblastoma by olaparib and assess the safety and tolerability of its combination with TMZ. METHODS: Preclinical pharmacokinetic studies evaluated olaparib tissue distribution in rats and tumor-bearing mice. Adult patients with recurrent glioblastoma received various doses and schedules of olaparib and low-dose TMZ in a 3 + 3 design. Suitable patients received olaparib prior to neurosurgical resection; olaparib concentrations in plasma, tumor core and tumor margin specimens were measured by mass spectrometry. A dose expansion cohort tested tolerability and efficacy of the recommended phase II dose (RP2D). Radiosensitizing effects of olaparib were measured by clonogenic survival in glioblastoma cell lines. RESULTS: Olaparib was a substrate for multidrug resistance protein 1 and showed no brain penetration in rats but was detected in orthotopic glioblastoma xenografts. Clinically, olaparib was detected in 71/71 tumor core specimens (27 patients; median, 496 nM) and 21/21 tumor margin specimens (9 patients; median, 512.3 nM). Olaparib exacerbated TMZ-related hematological toxicity, necessitating intermittent dosing. RP2D was olaparib 150 mg (3 days/week) with TMZ 75 mg/m2 daily for 42 days. Fourteen (36%) of 39 evaluable patients were progression free at 6 months. Olaparib radiosensitized 6 glioblastoma cell lines at clinically relevant concentrations of 100 and 500 nM. CONCLUSION: Olaparib reliably penetrates recurrent glioblastoma at radiosensitizing concentrations, supporting further clinical development and highlighting the need for better preclinical models.

Constant Degradation of the Androgen Receptor by MDM2 Conserves Prostate Cancer Stem Cell Integrity.

Prostate cancer stem cells (CSC) are implicated in tumor initiation, cancer progression, metastasis, and the development of therapeutic-resistant disease. It is well known that the bulk of prostate cancer cells express androgen receptor (AR) and that androgens are required for prostate cancer growth, progression, and emergence of castration-resistant disease. In contrast, the small subpopulation of self-renewing CSCs exhibits an AR-negative (AR-) signature. The mechanisms underlying the absence of AR are unknown. Using CSC-like cell models isolated from clinical biopsy tissues, we identify the E3 ligase MDM2 as a key regulator of prostate CSC integrity. First, unlike what has been reported for the bulk of AR+ tumor cells where MDM2 regulates the temporal expression of AR during transcriptional activity, MDM2 in CSCs promoted the constant ubiquitination and degradation of AR, resulting in sustained loss of total AR protein. Second, MDM2 promoted CSC self-renewal, the expression of stem cell factors, and CSC proliferation. Loss of MDM2 reversed these processes and induced expression of full-length AR (and not AR variants), terminal differentiation into luminal cells, and cell death. Selectively blocking MDM2-mediated activity in combination with androgen/AR-targeted therapy may offer a novel strategy for eliminating AR- CSCs in addition to the bulk of AR+ prostate cancer cells, decreasing metastatic tumor burden and inhibiting the emergence of therapeutic resistance.Significance: These findings provide a novel mechanistic aspect of prostate cancer cell stemness that advances our understanding of the diverse transcriptional activity that bypasses AR in contributing to therapeutic resistance, tumor progression, and metastasis.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/6/1124/F1.large.jpg.

Actions of endocrine-disrupting chemicals on stem/progenitor cells during development and disease.

Development and fate of the stem cell are regulated by extrinsic signals from the environment. Endocrine-disrupting chemicals which perturb hormonal signaling in utero and during early childhood may cause deregulation of multiple developmental processes, ranging from breakdown of stem cell niche architecture, developmental reprograming and altered stem cell fate to impaired organ and gonad development and sexual differentiation. Therefore, study of the environmental effects on stem cell integrity and normal development is a new and emerging focus for developmental biologists and cell toxicologists. When combined with new human and mouse stem cell-based models, stem cell differentiation dynamics can be studied in more biologically relevant ways. In this study, we review the current status of our understanding of the molecular mechanisms by which endocrine disruptors alter embryonic stem cell and adult stem/progenitor cell fate, organ development, cancer stem cell activity, and tumorigenesis.

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.

Estrogen-like disruptive effects of dietary exposure to bisphenol A or 17α-ethinyl estradiol in CD1 mice.

Bisphenol A (BPA) is an endocrine disrupting chemical that is ubiquitous in wild and built environments. Due to variability in study design, the disruptive effects of BPA have proven difficult to experimentally replicate. This study was designed to assess the disruptive actions of dietary BPA exposure, while carefully controlling for known confounders. Parental CD1 mice were acclimated to defined diet containing BPA (0.03, 0.3, 3, 30, or 300 ppm) or 17α-ethinyl estradiol (EE; 0.0001, 0.001, and 0.01 ppm) and bred to produce progeny (F1) that were maintained through adulthood on the same diet as the parents. In F1 females, uterine weights were increased in all EE and the 30-ppm BPA-exposure groups, demonstrating model sensitivity and estrogen-like actions of BPA. In BPA-exposed females, no treatment-related differences were observed in parental reproductive function, or in the timing of puberty and metabolic function in female offspring. In F1 males, modest changes in body weight, adiposity and glucose tolerance, consistent with improved metabolic function, were observed. Associated with increased prolactin and increased circulating testosterone levels, balanopreputial separation was accelerated by 0.03 and 3.0 ppm BPA and anogenital distance at postnatal day 21 was increased in males by 0.03 ppm BPA. Sperm counts were also increased with 3.0 ppm BPA exposures. Overall, BPA was found to have modest, sex specific endocrine disruptive effects on a variety of end points below the established no observed adverse effect level. The dose response characteristics for many of the effects were nonmonotonic and not predictable from high-dose extrapolations.

Inhibition of stathmin1 accelerates the metastatic process.

The oncoprotein stathmin 1 (STMN1) is upregulated in most, if not all, cancers of epithelial cell origin; therefore STMN1 is considered a target for cancer therapy. However, its role during metastasis has not been investigated. Here, we report for the first time that STMN1 strongly inhibits metastatic behavior in both normal epithelial and cancerous epithelial cells. Initially, loss-of-STMN1 compromises cell-cell adhesion. This is followed by epithelial-to-mesenchymal transition (EMT), increased cell migration, and metastasis via cooperative activation of p38 and through TGF-ß-independent and -dependent mechanisms. In contrast, expressing STMN1 restores cell-cell adhesion and reverses the metastatic cascade. Primary prostate epithelial cell cultures from benign to undifferentiated adenocarcinoma (UA) clinical biopsies show that EMT-like cells arise while the cancer is still organ-confined and that their emergence is tumor-stage specific. Furthermore, primary EMT-like cells exhibit metastatic behavior both in vitro and in vivo as compared with their non-EMT counterpart. These observations predict that using STMN1 as a generic therapeutic target might accelerate metastasis. Instead, there may be a tumor stage-specific window-of-opportunity in which conserving STMN1 expression is required to inhibit emergence of metastatic disease.

Androgen hormone action in prostatic carcinogenesis: stromal androgen receptors mediate prostate cancer progression, malignant transformation and metastasis.

It has been postulated that prostatic carcinogenesis is androgen dependent and that androgens mediate their effects primarily through epithelial cells; however, definitive proof of androgen hormone action in prostate cancer (PRCA) progression is lacking. Here we demonstrate through genetic loss of function experiments that PRCA progression is androgen dependent and that androgen dependency occurs via prostatic stromal androgen receptors (AR) but not epithelial AR. Utilizing tissue recombination models of prostatic carcinogenesis, loss of AR function was evaluated by surgical castration or genetic deletion. Loss of AR function prevented prostatic carcinogenesis, malignant transformation and metastasis. Tissue-specific evaluation of androgen hormone action demonstrated that epithelial AR was not necessary for PRCA progression, whereas stromal AR was essential for PRCA progression, malignant transformation and metastasis. Stromal AR was not necessary for prostatic maintenance, suggesting that the lack of cancer progression due to stromal AR deletion was not related to altered prostatic homeostasis. Gene expression analysis identified numerous androgen-regulated stromal factors. Four candidate stromal AR-regulated genes were secreted growth factors: fibroblast growth factors-2, -7, -10 and hepatocyte growth factor which were significantly affected by androgens and anti-androgens in stromal cells grown in vitro. These data support the concept that androgens are necessary for PRCA progression and that the androgen-regulated stromal microenvironment is essential to carcinogenesis, malignant transformation and metastasis and may serve as a potential target in the prevention of PRCA.

Regenerated luminal epithelial cells are derived from preexisting luminal epithelial cells in adult mouse prostate.

Determining the source of regenerated luminal epithelial cells in the adult prostate during androgen deprivation and replacement will provide insights into the origin of prostate cancer cells and their fate during androgen deprivation therapy. Prostate stem cells in the epithelial layer have been suggested to give rise to luminal epithelium. However, the extent of stem cell participation to prostate regrowth is not clear. In this report, using prostate-specific antigen-CreER(T2)-based genetic lineage marking/tracing in mice, preexisting luminal epithelial cells were shown to be a source of regenerated luminal epithelial cells in the adult prostate. Prostatic luminal epithelial cells could survive androgen deprivation and were capable of proliferating upon androgen replacement. Prostate cancer cells, typically exhibiting a luminal epithelial phenotype, may retain this intrinsic capability to survive and regenerate in response to changes in androgen signaling, providing part of the mechanism for the ultimate failure of androgen deprivation therapy in prostate cancer.

Thrombin expression in prostate: a novel finding.

INTRODUCTION: A variable repertoire of coagulation protein expression is observed in different cancers. We evaluated expression of thrombin in prostate tissue. METHODS: Detection of thrombin was performed using quantitative real-time PCR in fresh tissue and in situ hybridization (ISH) in archival prostate tissue and by immunohistochemistry of prostate tissue microarrays. RESULTS: (Pro)thrombin mRNA expression was detected in cancer tissue and localized to prostatic epithelium and stroma by ISH. Thrombin protein was detected in stroma of benign and malignant epithelium (p <.05). CONCLUSIONS: Prostate tissue is a rich reservoir of thrombin. This may have potential for developing antithrombin-based cancer therapy.

A novel in vitro assay of tumor-initiating cells in xenograft prostate tumors.

BACKGROUND: The field of prostate cancer has been stymied by the difficulty of cultivating patient-derived samples in the laboratory. In order to help circumvent this challenge, we sought to develop an in vitro assay of human prostate cancer initiation employing a prostate-associated mesenchymal feeder layer. METHODS: Rat seminal vesicle mesenchyme (rSVM) harvested from male neonatal rats was plated in 12-well plates and then irradiated with 30 Gy after approximately 75% confluence. Single-cell suspensions of two human non-adherent prostate cancer xenograft lines (TRPC and LAPC9) were then plated on irradiated rSVM. At 3-4 weeks, three-dimensional solid structures, termed glandoids, were harvested and analyzed or transplanted singly into the renal capsule of immunodeficient mice. Animals were assessed for tumor formation 8-12 weeks after engraftment. Finally, clonality assays were performed to determine whether glandoids usually arise from a single cell and are therefore clonal in origin. RESULTS: Glandoids form with reliable frequency (1/ approximately 300 plated cells), are constituted by relevant cell types (CK8+, CK5-, PSA+) and after implantation into immunocompromised mice, give rise to tumors that recapitulate original xenograft histology and cell composition; defining a glandoid as a tumor-initiating unit. In addition, assessment of red fluorescent protein (RFP)-labeled glandoids revealed either all red or non-red structures, with few areas of fusion, suggesting glandoids are clonal in origin. CONCLUSIONS: The above assay describes an adjunct technique to readily cultivate cells from prostate cancer xenografts in vitro and as such provides a platform on which tumor-initiating cell studies and high-throughput drug discovery may be performed.

Selective expression of CD44, a putative prostate cancer stem cell marker, in neuroendocrine tumor cells of human prostate cancer.

BACKGROUND: Hormonal therapy is effective for advanced prostate cancer (PC) but the disease often recurs and becomes hormone-refractory. It is hypothesized that a subpopulation of cancer cells, that is, cancer stem cells (CSCs), survives hormonal therapy and leads to tumor recurrence. CD44 expression was shown to identify tumor cells with CSC features. PC contains secretory type epithelial cells and a minor population of neuroendocrine cells. Neuroendocrine cells do not express androgen receptor and are quiescent, features associated with CSCs. The purpose of the study was to determine the expression of CD44 in human PC and its relationship to neuroendocrine tumor cells. METHODS: Immunohistochemistry and immunofluorescence were performed to study CD44 expression in PC cell lines, single cells from fresh PC tissue and archival tissue sections of PC. We then determined if CD44+ cells represent neuroendocrine tumor cells. RESULTS: In human PC cell lines, expression of CD44 is associated with cells of NE phenotype. In human PC tissues, NE tumor cells are virtually all positive for CD44 and CD44+ cells, excluding lymphocytes, are all NE tumor cells. CONCLUSIONS: Selective expression of the stem cell-associated marker CD44 in NE tumor cells of PC, in combination with their other known features, further supports the significance of such cells in therapy resistance and tumor recurrence.

Transcriptional profiling of inductive mesenchyme to identify molecules involved in prostate development and disease.

BACKGROUND: The mesenchymal compartment plays a key role in organogenesis, and cells within the mesenchyme/stroma are a source of potent molecules that control epithelia during development and tumorigenesis. We used serial analysis of gene expression (SAGE) to profile a key subset of prostatic mesenchyme that regulates prostate development and is enriched for growth-regulatory molecules. RESULTS: SAGE libraries were constructed from prostatic inductive mesenchyme and from the complete prostatic rudiment (including inductive mesenchyme, epithelium, and smooth muscle). By comparing these two SAGE libraries, we generated a list of 219 transcripts that were enriched or specific to inductive mesenchyme and that may act as mesenchymal regulators of organogenesis and tumorigenesis. We identified Scube1 as enriched in inductive mesenchyme from the list of 219 transcripts; also, quantitative RT-PCR and whole-mount in situ hybridization revealed Scube1 to exhibit a highly restricted expression pattern. The expression of Scube1 in a subset of mesenchymal cells suggests a role in prostatic induction and branching morphogenesis. Additionally, Scube1 transcripts were expressed in prostate cancer stromal cells, and were less abundant in cancer associated fibroblasts relative to matched normal prostate fibroblasts. CONCLUSION: The use of a precisely defined subset of cells and a back-comparison approach allowed us to identify rare mRNAs that could be overlooked using other approaches. We propose that Scube1 encodes a novel stromal molecule that is involved in prostate development and tumorigenesis.

Tissue-specific consequences of cyclin D1 overexpression in prostate cancer progression.

The cyclin D1 oncogene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the Rb protein and promotes progression through G(1) to S phase of the cell cycle. Several prostate cancer cell lines and a subset of primary prostate cancer samples have increased cyclin D1 protein expression. However, the relationship between cyclin D1 expression and prostate tumor progression has yet to be clearly characterized. This study examined the effects of manipulating cyclin D1 expression in either human prostatic epithelial or stromal cells using a tissue recombination model. The data showed that overexpression of cyclin D1 in the initiated BPH-1 cell line increased cell proliferation rate but did not elicit tumorigenicity in vivo. However, overexpression of cyclin D1 in normal prostate fibroblasts (NPF) that were subsequently recombined with BPH-1 did induce malignant transformation of the epithelial cells. The present study also showed that recombination of BPH-1 + cyclin D1-overexpressing fibroblasts (NPF(cyclin D1)) resulted in permanent malignant transformation of epithelial cells (BPH-1(NPF-cyclin D1) cells) similar to that seen with carcinoma-associated fibroblasts (CAF). Microarray analysis showed that the expression profiles between CAFs and NPF(cyclin D1) cells were highly concordant including cyclin D1 up-regulation. These data indicated that the tumor-promoting activity of cyclin D1 may be tissue specific.

Urothelial inhibition of transforming growth factor-beta in a bladder tissue recombination model.

PURPOSE: We examined the role of transforming growth factor-beta in urothelial and bladder development. Transforming growth factor-beta signaling was attenuated in the urothelial compartment and the subsequent effects were examined in a tissue recombination model. MATERIALS AND METHODS: Urothelium was cultured from adult rat bladders and transfected with control vector C7Delta or mutant DNIIR (dominant negative transforming growth factor-beta receptor II). Grafts were created by recombining transfected urothelium plus embryonic day 18 bladder mesenchyma and placed beneath the renal capsule of athymic mouse hosts. Grafts were harvested at 21 and 42 days. Final tissues were evaluated with staining and immunohistochemistry using hematoxylin and eosin, Gomori's trichrome strain, broad-spectrum uroplakin, smooth muscle actin-alpha, phosphorylated SMAD2 and Ki67 antigen. Bladder structures were defined as having smooth muscle, suburothelial connective tissue and mature urothelium expressing uroplakin. Urothelial compartment diameters were measured and subcategorized as small--0.10 to 0.40, medium--0.41 to 1.0 and large--greater than 1.1 mm. RESULTS: At 21 days 14 C7Delta control and 15 DNIIR grafts were evaluated. No bladder tissue was seen in the C7Delta grafts vs 49 in DNIIR tissue, including 30 small, 9 medium and 10 large tissues. At 42 days 14 C7Delta and 12 DNIIR grafts were evaluated. Six bladder structures (5 small and 1 medium) were seen in the C7Delta cohort vs 27 (14 small, 7 medium and 6 large) in the DNIIR group. Immunohistochemical detection of phosphorylated-SMAD2 was significantly attenuated in DNIIR tissue. In addition, Ki67 proliferative indexes were 4.0-fold higher in the DNIIR cohort compared to those in C7Delta tissues. CONCLUSIONS: We successfully observed that primary urothelium cultures can be genetically manipulated and recombined with undifferentiated mesenchyma to grow bladder tissue. By attenuating transforming growth factor-beta signaling in the urothelium superior bladder tissue growth occurred, suggesting that transforming growth factor-beta is a growth inhibitor in this organ system.

Cross-talk between paracrine-acting cytokine and chemokine pathways promotes malignancy in benign human prostatic epithelium.

The present study explores the mechanisms by which human prostatic carcinoma-associated fibroblasts (CAF) induce tumorigenesis in initiated but nonmalignant human prostatic epithelial cells (BPH-1). CAF express elevated levels of both transforming growth factor-beta1 (TGF-beta1) and stromal cell-derived factor-1 (SDF-1/CXCL12). TGF-beta inhibits the growth of BPH-1 cells in vitro, but was found to be necessary for the tumorigenic response to CAF. This counterintuitive result suggested that the TGF-beta signaling system was involved in other processes relating to tumorigenesis. The SDF-1 receptor, CXCR4, is expressed at low levels in benign prostate tissue and in BPH-1 cells in culture. However, CXCR4 levels increase during prostate cancer progression. CXCR4 was found to be induced and localized to the cell membrane in BPH1 cells by CAF-conditioned medium and by CAF cells in tissue recombinants. TGF-beta was both necessary and sufficient to allow the detection of membrane-localized CXCR4 in BPH1 cells. Suppression of epithelial cell CXCR4 expression abrogated the tumorigenic response to CAF. SDF-1, secreted by CAF, acts via the TGF-beta-regulated CXCR4 to activate Akt in the epithelial cells. This mechanism elicits tumorigenesis and obviates the growth-inhibitory effects of TGF-beta. Thus, tumor stroma can contribute to carcinogenesis through synergism between TGF-beta, SDF-1, and CXCR4. These experiments suggest mechanisms by which TGF-beta can shift its role from an inhibitor to a promoter of proliferation during tumor progression. Both the TGF-beta and SDF-1 pathways are targets of drug discovery efforts; these data suggest potential benefits in the cotargeting of these pathways.

Directed differentiation of embryonic stem cells into bladder tissue.

Manipulatable models of bladder development which interrogate specific pathways are badly needed. Such models will allow a systematic investigation of the multitude of pathologies which result from developmental defects of the urinary bladder. In the present communication, we describe a model in which mouse embryonic stem (ES) cells are directed to differentiate to form bladder tissue by specific interactions with fetal bladder mesenchyme. This model allows us to visualize the various stages in the differentiation of urothelium from ES cells, including the commitment to an endodermal cell lineage, with the temporal profile characterized by examining the induction of specific endodermal transcription factors (Foxa1 and Foxa2). In addition, final functional urothelial differentiation was characterized by examining uroplakin expression. It is well established that ES cells will spontaneously develop teratomas when grown within immunocompromised mouse hosts. We determined the specific mesenchymal to ES cell ratios necessary to dictate organ-specific differentiation while completely suppressing teratomatous growth. Embryonic mesenchyme is well established as an inductive tissue which dictates organ-specific programming of epithelial tissues. The present study demonstrates that embryonic bladder mesenchyme can also steer ES cells towards developing specific endodermal derived urothelium. These approaches allow us to capture specific stages of stem cell differentiation and to better define stem cell hierarchies.

Transforming growth factor-beta promotes invasion in tumorigenic but not in nontumorigenic human prostatic epithelial cells.

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor with actions that are dependent on circumstances, including dose, target cell type, and context. TGF-beta can elicit both growth-promoting and growth-suppressive activities. In normal tissues, TGF-beta generally acts to restrict growth and maintain differentiation. However, during tumorigenesis, changes in TGF-beta expression and cellular responses can promote tumorigenesis. The present study examines the effects of TGF-beta on the nontumorigenic human prostatic epithelial cell line BPH1 and on three derivative tumorigenic sublines BPH1(CAFTD)1, BPH1(CAFTD)3, and BPH1(CAFTD)5. The data show that TGF-beta has different effects on the nontumorigenic and tumorigenic cells. The nontumorigenic cells are growth inhibited by TGF-beta. In contrast, the tumorigenic sublines are not growth inhibited but instead undergo an epithelial to mesenchymal transformation (EMT) in response to TGF-beta. The tumorigenic lines show constitutively elevated levels of phosphorylated Akt, which modulates their response to TGF-beta by blocking Smad3 and p21 nuclear translocation. On TGF-beta stimulation of the tumorigenic sublines, the activated Akt allows the cell to escape cell cycle arrest. The phosphatidylinositol 3-kinase/Akt pathway is also involved in TGF-beta-induced EMT, defined here by induction of vimentin expression and enhanced cellular motility. In vivo, tumorigenic cells with constitutively active TGF-beta signaling show increased invasion with EMT, which express vimentin, located specifically at the invasive front of the tumor. These data indicate that following malignant transformation TGF-beta can play a direct role in promoting prostatic cancer and further that these responses are context specific in vivo.

Bladder tissue formation from cultured bladder urothelium.

Tissue recombination is a powerful method to evaluate the paracrine-signaling events that orchestrate the development of organs using the in vivo environment of a host rodent. Studies have reported the successful generation of primary cultures of rodent bladder urothelium, but none have reported their use to recapitulate bladder tissue with tissue recombination. We propose that primary cultured bladder urothelium, when recombined with inductive embryonic bladder mesenchyme, will form bladder tissue in a recombination model. Adult rat bladders were isolated and urothelium obtained. Sheets of bladder urothelium were re-suspended in collagen and maintained in tissue culture. After expansion (>20 passages), the urothelium was recombined with embryonic day-14 mouse bladder mesenchyme, then grafted beneath the renal capsule of immunocompromised mouse hosts. Grafts were harvested after 28 days. Control grafts were performed with bladder mesenchyme alone, cultured bladder urothelium alone, and collagen matrix alone. Final tissues were evaluated with staining and immunohistochemistry (H&E, Gomori's trichrome, broad-spectrum uroplakin, and smooth muscle actin alpha and gamma). Immunocytochemistry on cultured urothelium for broad-spectrum keratin, vimentin, and broad-spectrum uroplakin confirmed pure populations, void of mesenchymal contaminants. Staining of recombinant grafts demonstrated bladder tissue with mature urothelium and stromal differentiation. Control tissues were void of bladder tissue formation. We have successfully demonstrated that a chimeric bladder is formed from primary cultured bladder urothelium recombined with embryonic bladder mesenchyme. This is a powerful new tool for investigating the molecular mechanisms of bladder development and disease. Future applications may include the in vitro genetic manipulation of urothelium and examining those effects on growth and development in an in vivo environment.