RESUMEN
Here, we describe a novel pan-RAS inhibitor, ADT-007, that potently inhibited the growth of RAS mutant cancer cells irrespective of the RAS mutation or isozyme. RAS WT cancer cells with activated RAS from upstream mutations were equally sensitive. Conversely, cells from normal tissues or RAS WT cancer cells harboring downstream BRAF mutations were insensitive. Insensitivity to ADT-007 was attributed to low activated RAS levels and metabolic deactivation by UDP-glucuronosyltransferases expressed in normal cells but repressed in RAS mutant cancer cells. Cellular, biochemical, and biophysical experiments show ADT-007 binds nucleotide-free RAS to block GTP activation of RAS and MAPK/AKT signaling. Local administration of ADT-007 strongly inhibited tumor growth in syngeneic immune-competent and xenogeneic immune-deficient mouse models of colorectal and pancreatic cancer while activating innate and adaptive immunity in the tumor immune microenvironment. Oral administration of ADT-007 prodrug inhibited tumor growth, supporting further development of this novel class of pan-RAS inhibitors for treating RAS-driven cancers. SIGNIFICANCE: ADT-007 is a 1 st -in-class pan-RAS inhibitor with ultra-high potency and unique selectivity for cancer cells with mutant or activated RAS capable of circumventing resistance and activating antitumor immunity. Further development of ADT-007 analogs or prodrugs with oral bioavailability as a generalizable monotherapy or combined with immunotherapy is warranted.
RESUMEN
The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) involves a significant accumulation of fibroblasts as part of the host response to cancer. Using single-cell RNA sequencing, multiplex immunostaining, and several genetic mouse models, we identify carcinoma-associated fibroblasts (CAF) with opposing functions in PDAC progression. Depletion of fibroblast activation protein (FAP)+ CAFs results in increased survival, in contrast to depletion of alpha smooth muscle actin (αSMA)+ CAFs, which leads to decreased survival. Tumor-promoting FAP+ CAFs (TP-CAF) and tumor-restraining αSMA+ CAFs (TR-CAF) differentially regulate cancer-associated pathways and accumulation of regulatory T cells. Improved efficacy of gemcitabine is observed when IL6 is deleted from αSMA+ CAFs but not from FAP+ CAFs using dual-recombinase genetic PDAC models. Improved gemcitabine efficacy due to lack of IL6 synergizes with anti-PD-1 immunotherapy to significantly improve survival of PDAC mice. Our study identifies functional heterogeneity of CAFs in PDAC progression and their different roles in therapy response. SIGNIFICANCE: PDAC is associated with accumulation of dense stroma consisting of fibroblasts and extracellular matrix that regulate tumor progression. Here, we identify two distinct populations of fibroblasts with opposing roles in the progression and immune landscape of PDAC. Our findings demonstrate that fibroblasts are functionally diverse with therapeutic implications. This article is highlighted in the In This Issue feature, p. 1397.
Asunto(s)
Fibroblastos Asociados al Cáncer , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Fibroblastos Asociados al Cáncer/metabolismo , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Fibroblastos/metabolismo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Interleucina-6/uso terapéutico , Ratones , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is therapeutically recalcitrant and metastatic. Partial epithelial to mesenchymal transition (EMT) is associated with metastasis; however, a causal connection needs further unraveling. Here, we use single-cell RNA sequencing and genetic mouse models to identify the functional roles of partial EMT and epithelial stabilization in PDAC growth and metastasis. A global EMT expression signature identifies â¼50 cancer cell clusters spanning the epithelial-mesenchymal continuum in both human and murine PDACs. The combined genetic suppression of Snail and Twist results in PDAC epithelial stabilization and increased liver metastasis. Genetic deletion of Zeb1 in PDAC cells also leads to liver metastasis associated with cancer cell epithelial stabilization. We demonstrate that epithelial stabilization leads to the enhanced collective migration of cancer cells and modulation of the immune microenvironment, which likely contribute to efficient liver colonization. Our study provides insights into the diverse mechanisms of metastasis in pancreatic cancer and potential therapeutic targets.
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Carcinoma Ductal Pancreático/genética , Transición Epitelial-Mesenquimal/genética , Neoplasias Hepáticas/genética , Proteínas Nucleares/genética , Neoplasias Pancreáticas/genética , Factores de Transcripción de la Familia Snail/genética , Proteína 1 Relacionada con Twist/genética , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/genética , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/secundario , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Neoplasias Hepáticas/secundario , Masculino , Ratones , Proteínas Nucleares/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Análisis de la Célula Individual , Factores de Transcripción de la Familia Snail/metabolismo , Análisis de Supervivencia , Microambiente Tumoral/genética , Proteína 1 Relacionada con Twist/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/deficienciaRESUMEN
Epithelial-to-mesenchymal transition (EMT) is a recognized eukaryotic cell differentiation program that is also observed in association with invasive tumors. Partial EMT program in carcinomas imparts cancer cells with mesenchymal-like features and is proposed as essential for metastasis. Precise determination of the frequency of partial EMT program in cancer cells in tumors and its functional role in metastases needs unraveling. Here, we employed mesenchymal cell reporter mice driven by αSMA-Cre and Fsp1-Cre with genetically engineered mice that develop spontaneous pancreatic ductal adenocarcinoma (PDAC) to monitor partial EMT program. Both αSMA- and Fsp1-Cre-mediated partial EMT programs were observed in the primary tumors. The established metastases were primarily composed of cancer cells without evidence for a partial EMT program, as assessed by our fate mapping approach. In contrast, metastatic cancer cells exhibiting a partial EMT program were restricted to isolated single cancer cells or micrometastases (3-5 cancer cells). Collectively, our studies identify large metastatic nodules with preserved epithelial phenotype and potentially unravel a novel metastasis program in PDAC.
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Transición Epitelial-Mesenquimal , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/fisiopatología , Actinas/genética , Actinas/metabolismo , Animales , Modelos Animales de Enfermedad , Genes Reporteros , Integrasas/genética , Integrasas/metabolismo , Ratones , Metástasis de la Neoplasia/patología , Metástasis de la Neoplasia/fisiopatología , Proteína de Unión al Calcio S100A4/genética , Proteína de Unión al Calcio S100A4/metabolismoRESUMEN
Deregulated transforming growth factor-ß (TGFß) signaling is a common feature of many epithelial cancers. Deletion of TGFß receptor type 2 (TGFBR2) in fibroblast specific protein-1 (FSP1)-positive stromal cells induces squamous cell carcinoma in the murine forestomach, implicating fibroblast-derived hepatocyte growth factor (HGF) as the major driver of the epithelium carcinogenesis. Prior to cancer development, hyperproliferative FSP1+ fibroblasts lacking TGFBR2 accumulate in the forestomach, disrupting the regulatory signaling cross-talk with the forestomach epithelium. Here, concurrent loss in TGFBR2 and SMAD4 completely abrogates the development of forestomach cancer. Bone morphogenic protein-7 (BMP7) was highly upregulated in forestomach cancer tissue, activating Smad1/5/8 signaling, cell proliferation, and HGF production in TGFBR2-deficient FSP1+ fibroblasts. This stimulation by BMP7 was lost in the combined TGFBR2 and SMAD4 double knockout fibroblasts, which included a profound decrease in HGF expression. Thus, Smad4-mediated signaling is required to initiate epithelial carcinogenesis subsequent to TGFBR2 deletion in FSP1+ fibroblasts.Implications: These findings reveal a complex cross-talk between epithelial cells and the stroma, wherein Smad4 is required to elicit squamous cell carcinomas in the forestomach of mice with TGFBR2-deficient stromal cells. Mol Cancer Res; 16(10); 1568-78. ©2018 AACR.
Asunto(s)
Proteína Morfogenética Ósea 7/genética , Carcinoma de Células Escamosas/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Neoplasias Gástricas/genética , Animales , Carcinogénesis/genética , Carcinoma de Células Escamosas/patología , Proliferación Celular/genética , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Regulación Neoplásica de la Expresión Génica , Factor de Crecimiento de Hepatocito/genética , Humanos , Mucosa Intestinal/patología , Proteína de Unión al Calcio S100A4/genética , Proteína Smad4/genética , Neoplasias Gástricas/patología , Células del Estroma/metabolismo , Células del Estroma/patologíaRESUMEN
The exact nature and dynamics of pancreatic ductal adenocarcinoma (PDAC) immune composition remains largely unknown. Desmoplasia is suggested to polarize PDAC immunity. Therefore, a comprehensive evaluation of the composition and distribution of desmoplastic elements and T-cell infiltration is necessary to delineate their roles. Here we develop a novel computational imaging technology for the simultaneous evaluation of eight distinct markers, allowing for spatial analysis of distinct populations within the same section. We report a heterogeneous population of infiltrating T lymphocytes. Spatial distribution of cytotoxic T cells in proximity to cancer cells correlates with increased overall patient survival. Collagen-I and αSMA+ fibroblasts do not correlate with paucity in T-cell accumulation, suggesting that PDAC desmoplasia may not be a simple physical barrier. Further exploration of this technology may improve our understanding of how specific stromal composition could impact T-cell activity, with potential impact on the optimization of immune-modulatory therapies.
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Carcinoma Ductal Pancreático/patología , Linfocitos Infiltrantes de Tumor/patología , Neoplasias Pancreáticas/patología , Linfocitos T Citotóxicos/patología , Actinas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Carcinoma Ductal Pancreático/mortalidad , Colágeno Tipo I/metabolismo , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunohistoquímica/métodos , Masculino , Ratones , Persona de Mediana Edad , Neoplasias Pancreáticas/mortalidad , Pronóstico , Análisis Espacial , Análisis Espectral , Tasa de Supervivencia , Linfocitos T/patología , Análisis de Matrices TisularesRESUMEN
Acute kidney injury (AKI) and progressive chronic kidney disease (CKD) are intrinsically tied syndromes. In this regard, the acutely injured kidney often does not achieve its full regenerative capacity and AKI directly transitions into progressive CKD associated with tubulointerstitial fibrosis. Underlying mechanisms of such AKI-to-CKD progression are still incompletely understood and specific therapeutic interventions are still elusive. Because epigenetic modifications play a role in maintaining tissue fibrosis, we used a murine model of ischemia-reperfusion injury to determine whether aberrant promoter methylation of RASAL1 contributes causally to the switch between physiological regeneration and tubulointerstitial fibrogenesis, a hallmark of AKI-to-CKD progression. It is known that the antihypertensive drug hydralazine has demethylating activity, and that its optimum demethylating activity occurs at concentrations below blood pressure-lowering doses. Administration of low-dose hydralazine effectively induced expression of hydroxylase TET3, which catalyzed RASAL1 hydroxymethylation and subsequent RASAL1 promoter demethylation. Hydralazine-induced CpG promoter demethylation subsequently attenuated renal fibrosis and preserved excretory renal function independent of its blood pressure-lowering effects. In comparison, RASAL1 demethylation and inhibition of tubulointerstitial fibrosis was not detected upon administration of the angiotensin-converting enzyme inhibitor Ramipril in this model. Thus, RASAL1 promoter methylation and subsequent transcriptional RASAL1 suppression plays a causal role in AKI-to-CKD progression.
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Lesión Renal Aguda/tratamiento farmacológico , Proteínas de Unión al ADN/metabolismo , Proteínas Activadoras de GTPasa/genética , Hidralazina/uso terapéutico , Riñón/patología , Proteínas Proto-Oncogénicas/metabolismo , Insuficiencia Renal Crónica/prevención & control , Vasodilatadores/uso terapéutico , Inhibidores de la Enzima Convertidora de Angiotensina/farmacología , Animales , Islas de CpG , Metilación de ADN , Dioxigenasas , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Epigénesis Genética , Fibroblastos/metabolismo , Fibrosis , Humanos , Hidralazina/administración & dosificación , Riñón/citología , Riñón/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Cultivo Primario de Células , Regiones Promotoras Genéticas , Ramipril/farmacología , Eliminación Renal/efectos de los fármacos , Daño por Reperfusión/complicaciones , Vasodilatadores/administración & dosificaciónRESUMEN
Diagnosis of pancreatic ductal adenocarcinoma (PDAC) is associated with a dismal prognosis despite current best therapies; therefore new treatment strategies are urgently required. Numerous studies have suggested that epithelial-to-mesenchymal transition (EMT) contributes to early-stage dissemination of cancer cells and is pivotal for invasion and metastasis of PDAC. EMT is associated with phenotypic conversion of epithelial cells into mesenchymal-like cells in cell culture conditions, although such defined mesenchymal conversion (with spindle-shaped morphology) of epithelial cells in vivo is rare, with quasi-mesenchymal phenotypes occasionally observed in the tumour (partial EMT). Most studies exploring the functional role of EMT in tumours have depended on cell-culture-induced loss-of-function and gain-of-function experiments involving EMT-inducing transcription factors such as Twist, Snail and Zeb1 (refs 2, 3, 7-10). Therefore, the functional contribution of EMT to invasion and metastasis remains unclear, and genetically engineered mouse models to address a causal connection are lacking. Here we functionally probe the role of EMT in PDAC by generating mouse models of PDAC with deletion of Snail or Twist, two key transcription factors responsible for EMT. EMT suppression in the primary tumour does not alter the emergence of invasive PDAC, systemic dissemination or metastasis. Suppression of EMT leads to an increase in cancer cell proliferation with enhanced expression of nucleoside transporters in tumours, contributing to enhanced sensitivity to gemcitabine treatment and increased overall survival of mice. Collectively, our study suggests that Snail- or Twist-induced EMT is not rate-limiting for invasion and metastasis, but highlights the importance of combining EMT inhibition with chemotherapy for the treatment of pancreatic cancer.
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Resistencia a Antineoplásicos/efectos de los fármacos , Transición Epitelial-Mesenquimal , Metástasis de la Neoplasia/patología , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Proliferación Celular/efectos de los fármacos , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Masculino , Ratones , Invasividad Neoplásica/patología , Proteínas de Transporte de Nucleósidos/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Factores de Transcripción de la Familia Snail , Análisis de Supervivencia , Factores de Transcripción/deficiencia , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteína 1 Relacionada con Twist/deficiencia , Proteína 1 Relacionada con Twist/genética , Proteína 1 Relacionada con Twist/metabolismo , GemcitabinaRESUMEN
Kidney fibrosis is marked by an epithelial-to-mesenchymal transition (EMT) of tubular epithelial cells (TECs). Here we find that, during renal fibrosis, TECs acquire a partial EMT program during which they remain associated with their basement membrane and express markers of both epithelial and mesenchymal cells. The functional consequence of the EMT program during fibrotic injury is an arrest in the G2 phase of the cell cycle and lower expression of several solute and solvent transporters in TECs. We also found that transgenic expression of either Twist1 (encoding twist family bHLH transcription factor 1, known as Twist) or Snai1 (encoding snail family zinc finger 1, known as Snail) expression is sufficient to promote prolonged TGF-ß1-induced G2 arrest of TECs, limiting the cells' potential for repair and regeneration. In mouse models of experimentally induced renal fibrosis, conditional deletion of Twist1 or Snai1 in proximal TECs resulted in inhibition of the EMT program and the maintenance of TEC integrity, while also restoring cell proliferation, dedifferentiation-associated repair and regeneration of the kidney parenchyma and attenuating interstitial fibrosis. Thus, inhibition of the EMT program in TECs during chronic renal injury represents a potential anti-fibrosis therapy.
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Puntos de Control del Ciclo Celular , Transición Epitelial-Mesenquimal , Riñón/patología , Animales , Acuaporina 1/genética , Células Cultivadas , Fibrosis , Fase G2 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Proteína 1 de Transporte de Anión Orgánico/genéticaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is associated with marked fibrosis and stromal myofibroblasts, but their functional contribution remains unknown. Transgenic mice with the ability to delete αSMA(+) myofibroblasts in pancreatic cancer were generated. Depletion starting at either noninvasive precursor (pancreatic intraepithelial neoplasia) or the PDAC stage led to invasive, undifferentiated tumors with enhanced hypoxia, epithelial-to-mesenchymal transition, and cancer stem cells, with diminished animal survival. In PDAC patients, fewer myofibroblasts in their tumors also correlated with reduced survival. Suppressed immune surveillance with increased CD4(+)Foxp3(+) Tregs was observed in myofibroblast-depleted mouse tumors. Although myofibroblast-depleted tumors did not respond to gemcitabine, anti-CTLA4 immunotherapy reversed disease acceleration and prolonged animal survival. This study underscores the need for caution in targeting carcinoma-associated fibroblasts in PDAC.
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Carcinoma Ductal Pancreático/patología , Fibroblastos/patología , Fibrosis/patología , Neoplasias Pancreáticas/patología , Animales , Carcinoma Ductal Pancreático/inmunología , Modelos Animales de Enfermedad , Fibroblastos/inmunología , Fibrosis/inmunología , Humanos , Tolerancia Inmunológica , Ratones , Ratones Transgénicos , Neoplasias Pancreáticas/inmunología , Análisis de SupervivenciaRESUMEN
The metastatic spread of tumor epithelial cells accounts for over 90% of cancer-specific mortality; however, the molecular mechanisms that govern tumor spread and distant recolonization remain unclear. In this issue of JCI, Rokavec and colleagues shine light on this murky aspect of tumor biology by focusing through the lens of microenvironmental contributions, namely inflammation, as driving signals that set off a delicate, intracellular feedback loop among cytokine receptors, transcription factors and miRNAs. This study provides in vivo evidence and identifies molecular players behind the elusive switch that drives the epithelial-to-mesenchymal transition and the mesenchymal-to-epithelial transition.
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Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Femenino , Humanos , MasculinoRESUMEN
The reactive stroma surrounding tumor lesions performs critical roles ranging from supporting tumor cell proliferation to inducing tumorigenesis and metastasis. Therefore, it is critical to understand the cellular components and signaling control mechanisms that underlie the etiology of reactive stroma. Previous studies have individually implicated fibroblast growth factor receptor 1 (FGFR1) and canonical WNT/ß-catenin signaling in prostate cancer progression and the initiation and maintenance of a reactive stroma; however, both pathways are frequently found to be coactivated in cancer tissue. Using autochthonous transgenic mouse models for inducible FGFR1 (JOCK1) and prostate-specific and ubiquitously expressed inducible ß-catenin (Pro-Cat and Ubi-Cat, respectively) and bigenic crosses between these lines (Pro-Cat × JOCK1 and Ubi-Cat × JOCK1), we describe WNT-induced synergistic acceleration of FGFR1-driven adenocarcinoma, associated with a pronounced fibroblastic reactive stroma activation surrounding prostatic intraepithelial neoplasia (mPIN) lesions found both in in situ and reconstitution assays. Both mouse and human reactive stroma exhibited increased transforming growth factor-ß (TGF-ß) signaling adjacent to pathologic lesions likely contributing to invasion. Furthermore, elevated stromal TGF-ß signaling was associated with higher Gleason scores in archived human biopsies, mirroring murine patterns. Our findings establish the importance of the FGFR1-WNT-TGF-ß signaling axes as driving forces behind reactive stroma in aggressive prostate adenocarcinomas, deepening their relevance as therapeutic targets.
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Neoplasias de la Próstata/metabolismo , Receptor Tipo 1 de Factor de Crecimiento de Fibroblastos/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Wnt/metabolismo , Adenocarcinoma/metabolismo , Animales , Biopsia , Línea Celular Tumoral , Proliferación Celular , Transformación Celular Neoplásica , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Humanos , Inflamación , Masculino , Ratones , Ratones Desnudos , Ratones Transgénicos , Transducción de Señal , Especificidad de la Especie , Células del Estroma/metabolismoRESUMEN
Wnt signaling is crucial for a variety of biological processes, including body axis formation, planar polarity, stem cell maintenance and cellular differentiation. Therefore, targeted manipulation of Wnt signaling in vivo would be extremely useful. By applying chemical inducer of dimerization (CID) technology, we were able to modify the Wnt co-receptor, low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5), to generate the synthetic ligand inducible Wnt switch, iLRP5. We show that iLRP5 oligomerization results in its localization to disheveled-containing punctate structures and sequestration of scaffold protein Axin, leading to robust ß-catenin-mediated signaling. Moreover, we identify a novel LRP5 cytoplasmic domain critical for its intracellular localization and casein kinase 1-dependent ß-catenin signaling. Finally, by utilizing iLRP5 as a Wnt signaling switch, we generated the Ubiquitous Activator of ß-catenin (Ubi-Cat) transgenic mouse line. The Ubi-Cat line allows for nearly ubiquitous expression of iLRP5 under control of the H-2K(b) promoter. Activation of iLRP5 in isolated prostate basal epithelial stem cells resulted in expansion of p63(+) cells and development of hyperplasia in reconstituted murine prostate grafts. Independently, iLRP5 induction in adult prostate stroma enhanced prostate tissue regeneration. Moreover, induction of iLRP5 in male Ubi-Cat mice resulted in prostate tumor progression over several months from prostate hyperplasia to adenocarcinoma. We also investigated iLRP5 activation in Ubi-Cat-derived mammary cells, observing that prolonged activation results in mammary tumor formation. Thus, in two distinct experimental mouse models, activation of iLRP5 results in disruption of tissue homeostasis, demonstrating the utility of iLRP5 as a novel research tool for determining the outcome of Wnt activation in a precise spatially and temporally determined fashion.