Carcinoma-associated fibroblasts (CAFs) play a critical role in malignant progression. Loss of TGF-ϐ receptor II (TGFϐR2) in the prostate stroma is correlated with prostatic tumorigenesis. To determine the mechanisms by which stromal heterogeneity due to loss of TGFϐR2 might contribute to cancer progression, we attenuated TGF-ϐ signaling in a subpopulation of immortalized human prostate fibroblasts in a model of tumor progression. In a tissue recombination model, loss of TGFϐR2 function in 50% of the stromal cell population resulted in malignant transformation of the non-tumorigenic human prostate epithelial cell line BPH1. Mixing fibroblasts expressing the empty vector and dominant negative TGFϐR2 increased the expression of markers of myofibroblast differentiation [co-expression of vimentin and alpha smooth muscle actin (αSMA)] through elevation of TGF-ϐ1 and activation of the Akt pathway. In combination, these two populations of stromal cells recapitulated the tumor inductive activity of CAFs. TGFϐR2 activity in mixed stromal cell populations cultured in vitro caused secretion of factors that are known to promote tumor progression, including TGF-ϐ1, SDF1/CXCL12, and members of the FGF and BMP families. In vivo, tissue recombination of fibroblasts overexpressing TGF-ϐ1 and SDF1/CXCL12 not only induced transformation of BPH1 cells, but also promoted a robust growth of highly invasive cells, similar to effects produced by CAFs. While the precise nature and/or origin of the particular stromal cell populations in vivo remain unknown, these findings strongly link heterogeneity in TGF-ϐ signaling to tumor promotion by tumor stromal cells.

Tissue remodeling or regeneration is believed to initiate from multipotent stem and progenitor cells. We report here the establishment of two spontaneously immortalized adult non-tumorigenic human prostate epithelial cell lines, NHPrE1 and BHPrE1. NHPrE1 (CD133high/CD44high/OCT4high/PTENhigh) was characterized as a putative progenitor cell, and BHPrE1 (p63high/p53high/p21(WAF1)high/RBhigh) was characterized as a putative epithelial intermediate cell. Genomic analysis demonstrated an abnormal karyotype with genomic rearrangements including PTEN amplification in NHPrE1 and CTNNB1 (β-catenin) amplification in BHPrE1 cells. Embedded three-dimensional culture of NHPrE1 showed greater branching than BHPrE1. A tissue recombination-xenografting model was utilized to compare remodeling of human prostatic tissues in vivo. A series of tissue recombinants, made by mixing different ratios of human prostatic epithelial cells and inductive rat urogenital sinus mesenchyme, were grafted to the renal capsule of severe combined immunodeficient mice. Both cell lines were able to regenerate benign secretory ductal-acinar architecture in vivo, containing intact basal and luminal epithelial layers confirmed by the expression of appropriate CK profiles. Prostate-specific antigen, 15-lipoxygenase-2, androgen receptor, and NKX3.1 proteins were appropriately expressed in the regenerated epithelia. Regeneration of benign prostatic glandular structures could be achieved using as few as 10 NHPrE1 cells, whereas 200,000 BHPrE1 cells were required to achieve prostatic architecture. This suggests a greater proportion of progenitor/stem cells in NHPrE1 than in BHPrE1. These cell lines provide important data on progenitor and intermediate cell phenotypes and represent significant new tools for the elucidation of molecular mechanisms of human prostatic regeneration, pathogenesis, and carcinogenesis.

Key Points• We demonstrate that PT promotes ECFCs dysfunction by inducing stress-induced premature senescence.• Our data reveal that SIRT1 deficiency drives PT-ECFC senescence, and acts as a critical determinant of the PT-ECFC angiogenic defect.Epidemiological and experimental studies indicate that early vascular dysfunction occurs in low-birth-weight subjects, especially preterm (PT) infants. We recently reported impaired angiogenic activity of endothelial colony-forming cells (ECFCs) in this condition. We hypothesized that ECFC dysfunction in PT might result from premature senescence and investigated the underlying mechanisms. Compared with ECFCs from term neonates (n 5 18), ECFCs isolated from PT (n 5 29) display an accelerated senescence sustained by growth arrest and increased senescence-associated b-galactosidase activity. Increased p16INK4a expression, in the absence of telomere shortening, indicates that premature PT-ECFC aging results from stress-induced senescence. SIRT1 level, a nicotinamide adenine dinucleotidedependent deacetylase with anti-aging activities, is dramatically decreased in PT-ECFCs and correlated with gestational age. SIRT1 deficiency is subsequent to epigenetic silencing of its promoter. Transient SIRT1 overexpression or chemical induction by resveratrol treatment reverses senescence phenotype, and rescues in vitro PT-ECFC angiogenic defect in a SIRT1-dependent manner. SIRT1 overexpression also restores PT-ECFC capacity for neovessel formation in vivo. We thus demonstrate that decreased expression of SIRT1 drives accelerated senescence of PT-ECFCs, and acts as a critical determinant of the PT-ECFC angiogenic defect. These findings lay new grounds for understanding the increased cardiovascular risk in individuals born prematurely and open perspectives for therapeutic strategy. (Blood. 2014;123(13):2116-2126

SignificanceNanotechnology-based imaging is expected to bring breakthroughs in cancer diagnosis by improving imaging sensitivity and specificity while reducing toxicity. Here, we developed an innovative nanosystem for positron emission tomography (PET) imaging based on a self-assembling amphiphilic dendrimer. This dendrimer assembled spontaneously into uniform supramolecular nanomicelles with abundant PET reporting units on the surface. By harnessing both dendrimeric multivalence and the “enhanced permeation and retention” (EPR) effect, this dendrimer nanosystem effectively accumulated in tumors, leading to exceedingly sensitive and specific imaging of various tumors, especially those that are otherwise undetectable using the clinical gold reference 2-fluorodeoxyglucose ([18F]FDG). This study illustrates the power of nanotechnology based on self-assembling dendrimers to provide an effective platform for bioimaging and related biomedical applications.

BackgroundUremic toxicity may play a role in the elevated risk of developing cognitive impairment found among patients with CKD. Some uremic toxins, like indoxyl sulfate, are agonists of the transcription factor aryl hydrocarbon receptor (AhR), which is widely expressed in the central nervous system and which we previously identified as the receptor of indoxyl sulfate in endothelial cells.MethodsTo characterize involvement of uremic toxins in cerebral and neurobehavioral abnormalities in three rat models of CKD, we induced CKD in rats by an adenine-rich diet or by 5/6 nephrectomy; we also used AhR−/− knockout mice overloaded with indoxyl sulfate in drinking water. We assessed neurologic deficits by neurobehavioral tests and blood-brain barrier disruption by SPECT/CT imaging after injection of 99mTc-DTPA, an imaging marker of blood-brain barrier permeability.ResultsIn CKD rats, we found cognitive impairment in the novel object recognition test, the object location task, and social memory tests and an increase of blood-brain barrier permeability associated with renal dysfunction. We found a significant correlation between 99mTc-DTPA content in brain and both the discrimination index in the novel object recognition test and indoxyl sulfate concentrations in serum. When we added indoxyl sulfate to the drinking water of rats fed an adenine-rich diet, we found an increase in indoxyl sulfate concentrations in serum associated with a stronger impairment in cognition and a higher permeability of the blood-brain barrier. In addition, non-CKD AhR−/− knockout mice were protected against indoxyl sulfate–induced blood-brain barrier disruption and cognitive impairment.ConclusionsAhR activation by indoxyl sulfate, a uremic toxin, leads to blood-brain barrier disruption associated with cognitive impairment in animal models of CKD.

CD146 (MUC-18, MCAM) expression on cancer cells correlates with cancer progression and a bad prognosis in several tumors, including melanoma and pancreatic tumors. Deciphering the mechanism mediating the CD146 role in cancer is essential for generating new therapeutic strategies. We found that CD146 expression in cancer cells is associated with a secretion of soluble CD146 (sCD146) that constitutes an active player in tumor development. Indeed, sCD146 induces the overexpression of its binding protein, angiomotin, on both endothelial and cancer cells and promotes both paracrine effects on angiogenesis and autocrine effects on cancer cells proliferation and survival. These last effects are mediated in part through the induction and phosphorylation of c-myc in cancer cells. In mice models xenografted with human CD146-positive melanoma or pancreatic cancer cells, administration of a novel monoclonal antibody specifically targeting sCD146, but not its membrane form, successfully suppresses tumor vascularization and growth. Our findings demonstrate that sCD146 secreted by CD146-positive tumors mediates important pro-angiogenic and pro-tumoral effects. Targeting sCD146 with a novel neutralizing antibody could thus constitute an innovative therapeutic strategy for the treatment of CD146-positive tumors.

Peroxisome proliferator-activated receptor-gamma (PPARγ) regulates the interface between cellular lipid metabolism, redox status and organelle differentiation. Conditional prostatic epithelial knockout of PPARγ in mice resulted in focal hyperplasia which developed into mouse prostatic intraepithelial neoplasia (mPIN). The grade of PIN became more severe with time. Electron microscopy (EM) showed accumulated secondary lysosomes containing cellular organelles and debris suggestive of autophagy. Consistent with this analysis the autophagy marker LC3 was found to be upregulated in areas of PIN in PPARγ KO tissues. We selectively knocked down PPARγ2 isoform in wild-type mouse prostatic epithelial cells and examined the consequences of this in a tissue recombination model. Histopathologically grafted tissues resembled the conditional PPARγ KO mouse prostates. EM studies of PPARγ- and PPARγ2-deficient epithelial cells in vitro were suggestive of autophagy, consistent with the prostatic tissue analysis. This was confirmed by examining expression of beclin-1 and LC3. Gene expression profiling in PPARγ-/γ2-deficient cells indicated a major dysregulation of cell cycle control and metabolic signaling networks related to peroxisomal and lysosomal maturation, lipid oxidation and degradation. The putative autophagic phenotypes of PPARγ-deficient cells could be rescued by re-expression of either γ1 or γ2 isoform. We conclude that disruption of PPARγ signaling results in autophagy and oxidative stress during mPIN pathogenesis.

By using a retroviral infection strategy followed by tissue recombination we have created a model of human prostate cancer that demonstrates that the c-MYC gene is sufficient to induce carcinogenesis.