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.

Attenuated transforming growth factor beta signaling promotes metastasis in a model of HER2 mammary carcinogenesis.

INTRODUCTION: Transforming growth factor beta (TGFß) plays a major role in the regulation of tumor initiation, progression, and metastasis. It is depended on the type II TGFß receptor (TßRII) for signaling. Previously, we have shown that deletion of TßRII in mammary epithelial of MMTV-PyMT mice results in shortened tumor latency and increased lung metastases. However, active TGFß signaling increased the number of circulating tumor cells and metastases in MMTV-Neu mice. In the current study, we describe a newly discovered connection between attenuated TGFß signaling and human epidermal growth factor receptor 2 (HER2) signaling in mammary tumor progression. METHODS: All studies were performed on MMTV-Neu mice with and without dominant-negative TßRII (DNIIR) in mammary epithelium. Mammary tumors were analyzed by flow cytometry, immunohistochemistry, and immunofluorescence staining. The levels of secreted proteins were measured by enzyme-linked immunosorbent assay. Whole-lung mount staining was used to quantitate lung metastasis. The Cancer Genome Atlas (TCGA) datasets were used to determine the relevance of our findings to human breast cancer. RESULTS: Attenuated TGFß signaling led to a delay tumor onset, but increased the number of metastases in MMTVNeu/DNIIR mice. The DNIIR tumors were characterized by increased vasculogenesis, vessel leakage, and increased expression of vascular endothelial growth factor (VEGF). During DNIIR tumor progression, both the levels of CXCL1/5 and the number of CD11b+Gr1+ cells and T cells decreased. Analysis of TCGA datasets demonstrated a significant negative correlation between TGFBR2 and VEGF genes expression. Higher VEGFA expression correlated with shorter distant metastasis-free survival only in HER2+ patients with no differences in HER2-, estrogen receptor +/- or progesterone receptor +/- breast cancer patients. CONCLUSION: Our studies provide insights into a novel mechanism by which epithelial TGFß signaling modulates the tumor microenvironment, and by which it is involved in lung metastasis in HER2+ breast cancer patients. The effects of pharmacological targeting of the TGFß pathway in vivo during tumor progression remain controversial. The targeting of TGFß signaling should be a viable option, but because VEGF has a protumorigenic effect on HER2+ tumors, the targeting of this protein could be considered when it is associated with attenuated TGFß signaling.

ß-Catenin and K-RAS synergize to form primitive renal epithelial tumors with features of epithelial Wilms' tumors.

Wilms' tumor (WT) is the most common childhood renal cancer. Although mutations in known tumor-associated genes (WT1, WTX, and CATNB) occur only in a third of tumors, many tumors show evidence of activated ß-catenin-dependent Wnt signaling, but the molecular mechanism by which this occurs is unknown. A key obstacle to understanding the pathogenesis of WT is the paucity of mouse models that recapitulate its features in humans. Herein, we describe a transgenic mouse model of primitive renal epithelial neoplasms that have high penetrance and mimic the epithelial component of human WT. Introduction of a stabilizing ß-catenin mutation restricted to the kidney is sufficient to induce primitive renal epithelial tumors; however, when compounded with activation of K-RAS, the mice develop large, bilateral, metastatic, multifocal primitive renal epithelial tumors that have the histologic and staining characteristics of the epithelial component of human WT. These highly malignant tumors have increased activation of the phosphatidylinositol 3-kinase-AKT and extracellular signal-regulated kinase pathways, increased expression of total and nuclear ß-catenin, and increased downstream targets of this pathway, such as c-Myc and survivin. Thus, we developed a novel mouse model in which activated K-RAS synergizes with canonical Wnt/ß-catenin signaling to form metastatic primitive renal epithelial tumors that mimic the epithelial component of human WT.

CCL11 exacerbates colitis and inflammation-associated colon tumorigenesis.

CCL11, also known as eotaxin-1, is described as an eosinophil chemoattractant, which has been implicated in allergic and Th2 inflammatory diseases. We have reported that CCL11 is significantly increased in the serum of inflammatory bowel disease (IBD) patients, colonic eosinophils are increased and correlate with tissue CCL11 levels in ulcerative colitis patients, and CCL11 is increased in dextran sulfate sodium (DSS)-induced murine colitis. Here, we show that CCL11 is involved in the pathogenesis of DSS-induced colitis and in colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinogenesis (CAC). Ccl11-/- mice exposed to DSS then allowed to recover had significantly less body weight loss and a decrease in histologic injury versus wild-type (WT) mice. In the AOM-DSS model, Ccl11-/- mice exhibited decreased colonic tumor number and burden, histologic injury, and colonic eosinophil infiltration versus WT mice. Ccl11 is expressed by both colonic epithelial and lamina propria immune cells. Studies in bone marrow chimera mice revealed that hematopoietic- and epithelial-cell-derived CCL11 were both important for tumorigenesis in the AOM-DSS model. These findings indicate that CCL11 is important in the regulation of colitis and associated carcinogenesis and thus anti-CCL11 antibodies may be useful for treatment and cancer chemoprevention in IBD.

TRAIL and interferon-alpha act synergistically to induce renal cell carcinoma apoptosis.

PURPOSE: Despite modern targeted therapy metastatic renal cell carcinoma remains a deadly disease. Interferon-alpha (Calbiochem(R)) is currently used to treat this condition, mainly combined with the targeted anti-vascular endothelial growth factor antibody bevacizumab. TRAIL (Apo2 ligand/tumor necrosis factor related apoptosis inducing ligand) (Calbiochem) is a novel antineoplastic agent now in early phase clinical trials. Interferon-alpha and TRAIL can act synergistically to kill cancer cells but to our knowledge this has never been tested in the context of renal cell carcinoma. We hypothesized that TRAIL and interferon-alpha could synergistically induce apoptosis in renal cell carcinoma cells. MATERIALS AND METHODS: We treated renal cell carcinoma cell lines with recombinant TRAIL and/or interferon-alpha. Viability and apoptosis were assessed by MTS assay, flow cytometry and Western blot. Synergy was confirmed by isobologram. Interferon-alpha induced changes in renal cell carcinoma cell signaling were assessed by Western blot, flow cytometry and enzyme-linked immunosorbent assay. RESULTS: TRAIL and interferon-alpha acted synergistically to increase apoptotic cell death in renal cell carcinoma cells. Interferon-alpha treatment altered the ability of cells to activate extracellular signal-regulated kinase while inhibiting extracellular signal-regulated kinase with UO126 abrogated TRAIL and interferon-alpha apoptotic synergy. Interferon-alpha did not induce changes in TRAIL or death receptor expression, or change other known mediators of the intrinsic and extrinsic apoptotic cascade in the cells. CONCLUSIONS: TRAIL plus interferon-alpha synergistically induces apoptosis in renal cell carcinoma cells, which is due at least in part to interferon-alpha mediated changes in extracellular signal-regulated kinase activation. TRAIL and interferon-alpha combination therapy may be a novel approach to advanced renal cell carcinoma that warrants further testing in vivo.

Loss of solute carrier family 7 member 2 exacerbates inflammation-associated colon tumorigenesis.

Solute carrier family 7 member 2 (SLC7A2, also known as CAT2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in wound repair. We have reported that both SLC7A2 expression and L-Arg availability are decreased in colonic tissues from inflammatory bowel disease patients and that mice lacking Slc7a2 exhibit a more severe disease course when exposed to dextran sulfate sodium (DSS) compared to wild-type (WT) mice. Here, we present evidence that SLC7A2 plays a role in modulating colon tumorigenesis in the azoxymethane (AOM)-DSS model of colitis-associated carcinogenesis (CAC). SLC7A2 was localized predominantly to colonic epithelial cells in WT mice. Utilizing the AOM-DSS model, Slc7a2-/- mice had significantly increased tumor number, burden, and risk of high-grade dysplasia vs. WT mice. Tumors from Slc7a2-/- mice exhibited significantly increased levels of the proinflammatory cytokines/chemokines IL-1ß, CXCL1, CXCL5, IL-3, CXCL2, CCL3, and CCL4, but decreased levels of IL-4, CXCL9, and CXCL10 compared to tumors from WT mice. This was accompanied by a shift toward pro-tumorigenic M2 macrophage activation in Slc7a2-deficient mice, as marked by increased colonic CD11b+F4/80+ARG1+ cells with no alteration in CD11b+F4/80+NOS2+ cells by flow cytometry and immunofluorescence microscopy. The shift toward M2 macrophage activation was confirmed in bone marrow-derived macrophages from Slc7a2-/- mice. In bone marrow chimeras between Slc7a2-/- and WT mice, the recipient genotype drove the CAC phenotype, suggesting the importance of epithelial SLC7A2 in abrogating neoplastic risk. These data reveal that SLC7A2 has a significant role in the protection from CAC in the setting of chronic colitis, and suggest that the decreased SLC7A2 in inflammatory bowel disease (IBD) may contribute to CAC risk. Strategies to enhance L-Arg availability by supplementing L-Arg and/or increasing L-Arg uptake could represent a therapeutic approach in IBD to reduce the substantial long-term risk of colorectal carcinoma.

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.

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.

Airway bacteria drive a progressive COPD-like phenotype in mice with polymeric immunoglobulin receptor deficiency.

Mechanisms driving persistent airway inflammation in chronic obstructive pulmonary disease (COPD) are incompletely understood. As secretory immunoglobulin A (SIgA) deficiency in small airways has been reported in COPD patients, we hypothesized that immunobarrier dysfunction resulting from reduced SIgA contributes to chronic airway inflammation and disease progression. Here we show that polymeric immunoglobulin receptor-deficient (pIgR(-/-)) mice, which lack SIgA, spontaneously develop COPD-like pathology as they age. Progressive airway wall remodelling and emphysema in pIgR(-/-) mice are associated with an altered lung microbiome, bacterial invasion of the airway epithelium, NF-κB activation, leukocyte infiltration and increased expression of matrix metalloproteinase-12 and neutrophil elastase. Re-derivation of pIgR(-/-) mice in germ-free conditions or treatment with the anti-inflammatory phosphodiesterase-4 inhibitor roflumilast prevents COPD-like lung inflammation and remodelling. These findings show that pIgR/SIgA deficiency in the airways leads to persistent activation of innate immune responses to resident lung microbiota, driving progressive small airway remodelling and emphysema.

Deletion of TGF-ß signaling in myeloid cells enhances their anti-tumorigenic properties.

By crossing LysM-Cre and TGF-ß type II receptor (Tgfbr2) floxed mice we achieved specific deletion of Tgfbr2 in myeloid cells (Tgfbr2(MyeKO) mice). S.c.-injected (LLC, EL4-OVA) and implanted (MMTV-PyMT) carcinoma cells grow slower in Tgfbr2(MyeKO) mice. The number of CD45(+) cells in the tumor tissue was the same in both genotypes of mice, but upon analysis, the percentage of T cells (CD45(+)CD3(+)) in the KO mice was increased. By flow cytometry analysis, we did not detect any differences in the number and phenotype of TAMs, CD11b(+)Gr1(+), and DCs in Tgfbr2(MyeKO) compared with Tgfbr2(MyeWT) mice. ELISA and qRT-PCR data showed differences in myeloid cell functions. In Tgfbr2(MyeKO) TAMs, TNF-α secretion was increased, basal IL-6 secretion was down-regulated, TGF-ß did not induce any VEGF response, and there was decreased MMP9 and increased MMP2 and iNOS expression. TGF-ß did not have any effect on CD11b(+)Gr1(+) cells isolated from Tgfbr2(MyeKO) mice in the regulation of Arg, iNOS, VEGF, and CXCR4, and moreover, these cells have decreased suppressive activity relative to T cell proliferation. Also, we found that DCs from tumor tissue of Tgfbr2(MyeKO) mice have increased antigen-presented properties and an enhanced ability to stimulate antigen-specific T cell proliferation. We conclude that Tgfbr2 in myeloid cells has a negative role in the regulation of anti-tumorigenic functions of these cells, and deletion of this receptor decreases the suppressive function of CD11b(+)Gr1(+) cells and increases antigen-presenting properties of DCs and anti-tumorigenic properties of TAMs.

A sensitized screen of N-ethyl-N-nitrosourea-mutagenized mice identifies dominant mutants predisposed to diabetic nephropathy.

Diabetic nephropathy (DN) is a late diabetic complication that comprises progressively increasing albuminuria, declining GFR, and increased cardiovascular risk. Only a minority of patients with diabetes (25 to 40%) develop nephropathy, and there is evidence that heritable genetic factors predispose these "at-risk" individuals to DN. Comparing variability among inbred mouse strains with respect to a specific phenotype can model interhuman variability, and each strain represents a genetically homogeneous system with a defined risk for nephropathy. C57BL/6 mice, which are relatively resistant to DN, were mutagenized using N-ethyl-N-nitrosourea and screened for mutants that developed excess albuminuria on a sensitizing type 1 diabetic background contributed by the dominant Akita mutation in insulin-2 gene (Ins2(Akita)). Two of 375 diabetic G1 founders were found to exhibit albumin excretion rates persistently 10-fold greater than albumin excretion rates in nonmutagenized Ins2(Akita) controls. This albuminuria trait was heritable and transmitted to approximately 50% of Ins2(Akita) G2 and G3 progeny, consistent with a simple, dominantly inherited trait, but was never observed in nondiabetic offspring. During the course of 1 yr, albuminuric Ins2(Akita) G2 and G3 progeny developed reduced inulin clearance with elevated blood urea nitrogen and plasma creatinine. Glomerular histology revealed mesangial expansion, and glomerular basement membrane thickening as determined by electron microscopy was enhanced in diabetic mutant kidneys. Hereditary albuminuric N-ethyl-N-nitrosourea-induced mutants were redesignated as Nphrp1 (nephropathy1) and Nphrp2 (nephropathy2) mice for two generated lines. These novel mutants provide new, robust mouse models of DN and should help to elucidate the underlying genetic basis of predisposition to DN.

Activation of protein kinase C isozymes protects LLCPK1 cells from H2O2 induced necrotic cell death.

BACKGROUND/AIMS: We have previously reported that ischemia/reperfusion injury (IRI) to the kidney leads to induced expression of RACK1 and changes in the level of expression and subcellular distribution of PKC isozymes alpha, betaII and zeta. In order to further define the role of PKC isozymes in IRI we investigated the effect of activation or inhibition of the isozymes on cytotoxicity mediated by H(2)O(2) in LLCPK(1) cells. METHODS: Cytotoxicity was analyzed by Trypan blue assay and LDH release assay. Translocation of PKC isozymes postinjury in LLCPK1 cells was analyzed by immunostaining and Western blot analysis. RESULTS: Western blot analysis showed that the expression of PKC-alpha was up-regulated in a triphasic pattern with the initial induction within the first 10 min of injury followed by higher levels of expression at 2 and 24 h postinjury. The expression of PKC-zeta was highly induced within the first 15 min of injury but its expression was down-regulated to that of normal levels by 30 min postinjury. Immunocytochemistry showed that both PKC-alpha and PKC-zeta translocated to the nucleus and perinuclear region during H(2)O(2) treatment. Following injury, PKC-alpha expression was localized to the nuclear membrane at earlier time points but a translocation to the nucleus occurred at later time points. PKC-zeta translocated to nucleus at 30 minutes post injury and relocated back to the nuclear membrane at later time points. CONCLUSION: These data suggest that activation of PKC-alpha and PKC-zeta is involved in the H(2)O(2) induced injury of LLCPK1 cells.