Inhibition of Transforming Growth Factor-ß Improves Primary Renal Tubule Cell Differentiation in Long-Term Culture.

Patient-oriented applications of cell culture include cell therapy of organ failure like chronic renal failure. Clinical deployment of a cell-based device for artificial renal replacement requires qualitative and quantitative fidelity of a cultured cell to its in vivo counterpart. Active specific apicobasal ion transport reabsorbs 90-99% of the filtered load of salt and water in the kidney. In a bioengineered kidney, tubular transport concentrates wastes and eliminates the need for hemodialysis, but renal tubule cells in culture transport little or no salt and water due to dedifferentiation that mammalian cells undergo in vitro thereby losing important cell-type specific functions. We previously identified transforming growth factor-ß (TGF-ß) as a signaling pathway necessary for in vitro differentiation of renal tubule cells. Inhibition of TGF-ß receptor-1 led to active and inhibitable electrolyte and water transport by primary human renal tubule epithelial cells in vitro. Addition of metformin increased transport, in the context of a transient effect on 5'-AMP-activated kinase phosphorylation. These data motivated us to examine whether increased transport was an idiosyncratic effect of SB431542, probe pathways downstream of TGF-ß receptors possibly responsible for the improved differentiation, evaluate whether TGF-ß inhibition induced a range of differentiated tubule functions, and to explore crosstalk between the effects of SB431542 and metformin. In this study, we use multiple small-molecule inhibitors of canonical and noncanonical pathways to confirm that inhibition of canonical TGF-ß signaling caused the increased apicobasal transport. Hallmarks of proximal tubule cell function, including sodium reabsorption, para-amino hippurate excretion, and glucose uptake increased with TGF-ß inhibition, and the specificity of the response was shown using inhibitors of each transport protein. We did not find any evidence of crosstalk between metformin and SB431542. These data suggest that the TGF-ß signaling pathway governs multiple features of differentiation in renal proximal tubule cells in vitro. Inhibition of TGF-ß by pharmacologic or genome engineering approaches may be a viable approach to enhancing differentiated function of tubule cells in vitro. Impact statement Cell therapy of renal failure requires qualitative and quantitative fidelity between in vitro and in vivo phenotypes, which has been elusive. We show that control of transforming growth factor-ß signaling can promote differentiation of renal tubule cells grown in artificial environments. This is a key enabling step for cell therapy of renal failure.

Substrate Elasticity Governs Differentiation of Renal Tubule Cells in Prolonged Culture.

IMPACT STATEMENT: Successful clinical tissue engineering requires functional fidelity of the cultured cell to its in vivo counterpart, but this has been elusive in renal tissue engineering. Typically, renal proximal tubule cells in culture have a flattened morphology and do not express key transporters essential to their function. In this article, we show for the first time that in vitro substrate mechanical properties dictate differentiation of cultured renal proximal tubule cells. Remarkably, this effect was only discernable after 4 weeks in culture, longer than usually reported for this cell type. These results demonstrate a new tunable parameter to optimize cell differentiation in renal tissue engineering.

Pharmacologic Inhibition of ß-Catenin With Pyrvinium Inhibits Murine and Human Models of Wilms Tumor.

Wilms tumor (WT) is the most common renal malignancy in children and the fourth most common pediatric solid malignancy in the US. Although the mechanisms underlying the WT biology are complex, these tumors most often demonstrate activation of the canonical Wnt/ß-catenin pathway. We and others have shown that constitutive activation of ß-catenin restricted to the renal epithelium is sufficient to induce primitive renal epithelial tumors, which resemble human WT. Here we demonstrate that pharmacologic inhibition of ß-catenin gene transcription with pyrvinium inhibits tumor growth and metastatic progression in a murine model of WT. Cellular invasion is significantly inhibited in both murine WT-like and human WT cells and is accompanied by downregulation of the oncogenes Myc and Birc5 (survivin). Our studies provide proof of the concept that the canonical Wnt/ß-catenin pathway may be a novel therapeutic target in the management of WT.

Glyoxalase 2 drives tumorigenesis in human prostate cells in a mechanism involving androgen receptor and p53-p21 axis.

Glyoxalase 2 (Glo2), a metabolic enzyme, is overexpressed in some human cancers which suggests this enzyme may play a role in human tumorigenesis. In prostate cancer (PCa), the role of Glo2 has been scarcely investigated and there are no studies addressing a causative involvement of this protein in this neoplasia. Here, we examined the immunohistochemical profile of Glo2 in human PCa and benign adjacent tissues and investigated Glo2 involvement in PCa development in human prostate cell lines. PCa and matched adjacent normal tissues were obtained from paraffin sections of primary PCa from 20 patients who had undergone radical prostatectomy. Histopathological diagnosis was confirmed for each sample. Glo2 expression analysis was performed by immunohistochemistry in prostate tissues, and by qRT-PCR and immunoblotting in prostate cell lines. The causative and mechanistic role of Glo2 in prostate tumorigenesis was demonstrated by Glo2 ectopic expression/silencing and employing specific activators/inhibitors. Our results showed that Glo2 was selectively expressed in PCa but not in the luminal compartment of the adjacent benign epithelium consistently in all the examined 20 cases. Glo2 expression in PCa was dependent on androgen receptor (AR) and was aimed at stimulating cell proliferation and eluding apoptosis through a mechanism involving the p53-p21 axis. Glo2 was intensely expressed in the basal cells of benign glands but was not involved in PCa genesis. Our results demonstrate for the first time that Glo2 drives prostate tumorigenesis and suggest that it may represent a novel adjuvant marker in the pathological diagnosis of early PCa.

Functional KRAS mutations and a potential role for PI3K/AKT activation in Wilms tumors.

Wilms tumor (WT) is the most common renal neoplasm of childhood and affects 1 in 10 000 children aged less than 15 years. These embryonal tumors are thought to arise from primitive nephrogenic rests that derive from the metanephric mesenchyme during kidney development and are characterized partly by increased Wnt/ß-catenin signaling. We previously showed that coordinate activation of Ras and ß-catenin accelerates the growth and metastatic progression of a murine WT model. Here, we show that activating KRAS mutations can be found in human WT. In addition, high levels of phosphorylated AKT are present in the majority of WT. We further show in a mouse model and in renal epithelial cells that Ras cooperates with ß-catenin to drive metastatic disease progression and promotes in vitro tumor cell growth, migration, and colony formation in soft agar. Cellular transformation and metastatic disease progression of WT cells are in part dependent on PI3K/AKT activation and are inhibited via pharmacological inhibition of this pathway. Our studies suggest both KRAS mutations and AKT activation are present in WT and may represent novel therapeutic targets for this disease.

TPX2 as a prognostic indicator and potential therapeutic target in clear cell renal cell carcinoma.

OBJECTIVES: Our aims were to determine if targeting protein for Xklp2 (TPX2) is correlated with clear cell renal cell carcinoma (ccRCC) histology and oncologic outcomes using The Cancer Genome Atlas (TCGA) and an institutional tissue microarray (TMA). METHODS: Clinicopathological data obtained from the TCGA consisted of 415 samples diagnosed with ccRCC. A TMA was constructed from tumors of 207 patients who underwent radical nephrectomy for ccRCC. TPX2 expression by immunohistochemistry on TMA was assessed by a genitourinary pathologist. Clinical data were extracted and linked to TMA cores. TPX2 and Aurora-A mRNA coexpression were evaluated in the TCGA cohort. Overall survival (OS), cancer-specific survival, and recurrence-free survival (RFS) were analyzed using the Kaplan-Meier method and log-rank statistics. Univariate and multivariate analyses were conducted using Cox proportional hazard models. RESULTS: Median follow-up time for the TCGA cohort was 3.07 years. Aurora-A and TPX2 mRNA coexpression were significantly correlated (Pearson correlation = 0.918). High TPX2 mRNA expression was associated with advanced stage, metastasis, poor OS, and RFS. Median follow-up time for the TMA cohort was 5.3 years. Elevated TPX2 protein expression, defined as greater than 75th percentile staining intensity, was identified in 47/207 (22.7%) patients. Increased TPX2 immunostaining was associated with poor OS (P = 0.0327, 53% 5-year mortality), cancer-specific survival (P<0.01, 47.8% 5-year cancer-specific mortality), RFS (P = 0.0313, 73.6%, 5-year recurrence rate), grade, T stage, and metastasis. Multivariate analysis demonstrated elevated expression served as an independent predictor of RFS (hazard ratio = 3.62 (1.13-11.55), P = 0.029). CONCLUSIONS: We show TPX2, a regulator of Aurora-A, is associated with high grade and stage of ccRCC, and is an independent predictor of recurrence. Future studies are warranted testing its role in ccRCC biology, and its potential as a therapeutic target.

Glyoxalase 2 Is Involved in Human Prostate Cancer Progression as Part of a Mechanism Driven By PTEN/PI3K/AKT/mTOR Signaling With Involvement of PKM2 and ERα.

BACKGROUND: Glyoxalase 2 (Glo2), together with glyoxalase 1 (Glo1), forms the main scavenging system of methylglyoxal, a potent pro-apoptotic agent mainly generated by glycolysis. An increased rate of glycolysis is a well known signature of cancer cells. As a survival strategy, Glo1 is overexpressed in many human malignant cells, including prostate cancer (PCa), where it plays a crucial role in progression. No information is available on the role of Glo2 in the same ambit. PCa is the most common malignancy affecting men in the western world. Progression to a lethal hormone-refractory PCa represents the major concern in this pathology. Therefore, a deeper understanding of the molecular mechanisms underlying PCa invasiveness and metastasis is urgently needed in order to develop novel therapeutic targets for this incurable state of the malignancy. METHODS: Glo2 and Glo1 expression was examined in clinical samples of PCa by immunohistochemistry and in different PCa cell models by western blotting and quantitative real-time polymerase chain reaction. Gene silencing/overexpression and scavenging/inhibitory agents were used for functional analyses. RESULTS: We demonstrated that Glo2, together with Glo1, represents a novel mechanism in PCa progression as part of a pathway driven by PTEN/PI3K/AKT/mTOR signaling with involvement of PKM2 and ERα. Importantly, Glo1/Glo2 silencing did not alter the behavior of benign cells. CONCLUSIONS: Targeting glyoxalases metabolic pathway may represent a strategy to selectively inhibit advanced PCa. Prostate 77:196-210, 2017. © 2016 Wiley Periodicals, Inc.

A Murine Model of K-RAS and ß-Catenin Induced Renal Tumors Expresses High Levels of E2F1 and Resembles Human Wilms Tumor.

PURPOSE: Wilms tumor is the most common renal neoplasm of childhood. We previously found that restricted activation of the WNT/ß-catenin pathway in renal epithelium late in kidney development is sufficient to induce small primitive neoplasms with features of epithelial Wilms tumor. Metastatic disease progression required simultaneous addition of an activating mutation of the oncogene K-RAS. We sought to define the molecular pathways activated in this process and their relationship to human renal malignancies. MATERIALS AND METHODS: Affymetrix® expression microarray data from murine kidneys with activation of K-ras and/or Ctnnb1 (ß-catenin) restricted to renal epithelium were analyzed and compared to publicly available expression data on normal and neoplastic human renal tissue. Target genes were verified by immunoblot and immunohistochemistry. RESULTS: Mouse kidney tumors with activation of K-ras and Ctnnb1, and human renal malignancies had similar mRNA expression signatures and were associated with activation of networks centered on ß-catenin and TP53. Up-regulation of WNT/ß-catenin targets (MYC, Survivin, FOXA2, Axin2 and Cyclin D1) was confirmed by immunoblot. K-RAS/ß-catenin murine kidney tumors were more similar to human Wilms tumor than to other renal malignancies and demonstrated activation of a TP53 dependent network of genes, including the transcription factor E2F1. Up-regulation of E2F1 was confirmed in murine and human Wilms tumor samples. CONCLUSIONS: Simultaneous activation of K-RAS and ß-catenin in embryonic renal epithelium leads to neoplasms similar to human Wilms tumor and associated with activation of TP53 and up-regulation of E2F1. Further studies are warranted to evaluate the role of TP53 and E2F1 in human Wilms tumor.

Hypertension is a major contributor to 20-hydroxyeicosatetraenoic acid-mediated kidney injury in diabetic nephropathy.

In the kidney, 20-hydroxyeicosatetraenoic acid (20-HETE) is a primary cytochrome P450 4 (Cyp4)-derived eicosanoid that enhances vasoconstriction of renal vessels and induces hypertension, renal tubular cell hypertrophy, and podocyte apoptosis. Hypertension and podocyte injury contribute to diabetic nephropathy and are strong predictors of disease progression. In this study, we defined the mechanisms whereby 20-HETE affects the progression of diabetic nephropathy. We used Cyp4a14KO male mice that exhibit androgen-sensitive hypertension due to increased Cyp4a12-mediated 20-HETE production. We show that, upon induction of diabetes type 1 via streptozotocin injection, Cyp4a14KO male mice developed worse renal disease than streptozotocin-treated wild-type mice, characterized by increased albuminuria, mesangial expansion, glomerular matrix deposition, and thickness of the glomerular basement membranes. Castration blunted androgen-mediated Cyp4a12 synthesis and 20-HETE production, normalized BP, and ameliorated renal damage in diabetic Cyp4a14KO mice. Notably, treatment with a 20-HETE antagonist or agents that normalized BP without affecting Cyp4a12 expression and 20-HETE biosynthesis also ameliorated diabetes-mediated renal damage and albuminuria in Cyp4a14KO male mice. Taken together, these results suggest that hypertension is the major contributor to 20-HETE-driven diabetes-mediated kidney injury.

Deficiency in metabolic regulators PPARγ and PTEN cooperates to drive keratinizing squamous metaplasia in novel models of human tissue regeneration.

Hindgut-derived endoderm can differentiate into rectal, prostatic, and bladder phenotypes. Stromal-epithelial interactions are crucial for this development; however, the precise mechanisms by which epithelium responds to stromal cues remain unknown. We have previously reported ectopic expression of peroxisome proliferator-activated receptor-γ2 (PPARγ2) increased androgen receptor expression and promoted differentiation of mouse prostate epithelium. PPARγ is also implicated in urothelial differentiation. Herein we demonstrate that knockdown of PPARγ2 in benign human prostate epithelial cells (BHPrEs) promotes urothelial transdifferentiation. Furthermore, in vitro and in vivo heterotypic tissue regeneration models with embryonic bladder mesenchyme promoted urothelial differentiation of PPARγ2-deficient BHPrE cells, and deficiency of both PPARγ isoforms 1 and 2 arrested differentiation. Because PTEN deficiency is cooperative in urothelial pathogenesis, we engineered BHPrE cells with combined knockdown of PPARγ and PTEN and performed heterotypic recombination experiments using embryonic bladder mesenchyme. Whereas PTEN deficiency alone induced latent squamous differentiation in BHPrE cells, combined PPARγ and PTEN deficiency accelerated the development of keratinizing squamous metaplasia (KSM). We further confirmed via immunohistochemistry that gene expression changes in metaplastic recombinants reflected human urothelium undergoing KSM. In summary, these data suggest that PPARγ isoform expression provides a molecular basis for observations that adult human epithelium can be transdifferentiated on the basis of heterotypic mesenchymal induction. These data also implicate PPARγ and PTEN inactivation in the development of KSM.

Androgen regulated genes in human prostate xenografts in mice: relation to BPH and prostate cancer.

Benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) are linked to aging and the presence of androgens, suggesting that androgen regulated genes play a major role in these common diseases. Androgen regulation of prostate growth and development depends on the presence of intact epithelial-stromal interactions. Further, the prostatic stroma is implicated in BPH. This suggests that epithelial cell lines are inadequate to identify androgen regulated genes that could contribute to BPH and CaP and which could serve as potential clinical biomarkers. In this study, we used a human prostate xenograft model to define a profile of genes regulated in vivo by androgens, with an emphasis on identifying candidate biomarkers. Benign transition zone (TZ) human prostate tissue from radical prostatectomies was grafted to the sub-renal capsule site of intact or castrated male immunodeficient mice, followed by the removal or addition of androgens, respectively. Microarray analysis of RNA from these tissues was used to identify genes that were; 1) highly expressed in prostate, 2) had significant expression changes in response to androgens, and, 3) encode extracellular proteins. A total of 95 genes meeting these criteria were selected for analysis and validation of expression in patient prostate tissues using quantitative real-time PCR. Expression levels of these genes were measured in pooled RNAs from human prostate tissues with varying severity of BPH pathologic changes and CaP of varying Gleason score. A number of androgen regulated genes were identified. Additionally, a subset of these genes were over-expressed in RNA from clinical BPH tissues, and the levels of many were found to correlate with disease status. Our results demonstrate the feasibility, and some of the problems, of using a mouse xenograft model to characterize the androgen regulated expression profiles of intact human prostate tissues.

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.

Unopposed c-MYC expression in benign prostatic epithelium causes a cancer phenotype.

BACKGROUND: We have sought to develop a new in vivo model of prostate carcinogenesis using human prostatic epithelial cell cultures. Human prostate cancers frequently display DNA amplification in the 8q24 amplicon, which leads to an increase in the copy number of the c-MYC gene, a finding that suggests a role for c-MYC in human prostate carcinogenesis. In addition overexpression of c-MYC in transgenic mouse models results in prostatic carcinogenesis. METHODS: We took advantage of the ability of retroviruses to integrate foreign DNA into human prostatic epithelium (huPrE) to generate cell lines that overexpress the c-MYC protooncogene. These cells were recombined with inductive rat urogenital sinus mesenchyme and grafted beneath the renal capsule of immunocompromised rodent hosts. RESULTS: The resultant tissue displayed a phenotype consistent with a poorly differentiated human prostatic adenocarcinoma. The tumors were rapidly growing with a high proliferative index. The neoplastic cells in the tumor expressed both androgen receptors (AR) and prostate-specific antigen (PSA), both characteristic markers of human prostate cancers. Microarray analysis of human prostatic epithelial cells overexpression c-MYC identified a large number of differentially expressed genes some of which have been suggested to characterize a subset of human cancers that have myc overexpression. Specific examples were confirmed by Western blot analysis and include upregulation of c-Myb and decreased expression of PTEN. Control grafts using either uninfected huPrE or using huPrE cells infected using an empty vector expressing a green fluorescent protein tag gave rise to well differentiated benign prostatic glandular ducts. CONCLUSIONS: 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.