August Vidal

Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis

Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC-Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate nontumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies.

Mammalian Target of Rapamycin Pathway Blockade Slows Progression of Diabetic Kidney Disease in Rats

Recent data suggest that the phosphatidylinositol 3-kinase (PI3-K)/Akt/mammalian target of rapamycin (mTOR) pathway is important in diabetic nephropathy. The effect of mTOR blockade by sirolimus (SRL) in diabetic kidney disease in rats was investigated. Diabetes was induced by streptozotocin in male Sprague-Dawley rats. Sixteen weeks later, diabetic animals were divided into the following groups: diabetes (D; n = 8), diabetes + SRL at 1 mg/kg per d, SRL trough level 2.3 +/- 0.25 ng/ml (D+SRL; n = 7); and diabetes + normoglycemia maintained by insulin implants (D+NG; n = 5). There was an age-matched nondiabetic group (ND; n = 6). All animals were followed for 4 wk. The D group showed glomerular hypertrophy (mean glomerular volume 5.0 +/- 0.4 in D versus 3.3 +/- 0.2 10(6) mu(3) in ND; P < 0.05) without renal hyperplasia (calculated by reverse transcription-PCR of proliferative cell nuclear antigen) and albuminuria (29 +/- 4 in D versus 1.4 +/- 1.5 mg/24 h in ND; P < 0.05). Both D+NG and D+SRL groups had a significant reduction of albuminuria, although glomerular hypertrophy was still present. SRL treatment did not modify the number of infiltrating renal ED1(+) cells. Diabetic animals had greater expression of p-Akt and mTOR, unlike ND rats. NG and SRL treatment reduced p-Akt and normalized mTOR. It is interesting that D+SRL was associated with a significant reduction of renal TGF-beta1 and glomerular connective tissue growth factor. SRL treatment reduced glomerular alpha-smooth muscle actin overexpression and reduced significantly the mesangial matrix accumulation that is characteristic of diabetic nephropathy. In conclusion, mTOR blockade by low-dose SRL has a beneficial effect in diabetic kidney disease, suggesting that the mTOR pathway has an important pathogenic role in diabetic nephropathy.

Small molecule enoxacin is a cancer-specific growth inhibitor that acts by enhancing TAR RNA-binding protein 2-mediated microRNA processing

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression at the posttranscriptional level and are critical for many cellular pathways. The disruption of miRNAs and their processing machineries also contributes to the development of human tumors. A common scenario for miRNA expression in carcinogenesis is emerging that shows that impaired miRNA production and/or down-regulation of these transcripts occurs in many neoplasms. Several of these lost miRNAs have tumor-suppressor features, so strategies to restore their expression globally in malignancies would be a welcome addition to the current therapeutic arsenal against cancer. Herein, we show that the small molecule enoxacin, a fluoroquinolone used as an antibacterial compound, enhances the production of miRNAs with tumor suppressor functions by binding to the miRNA biosynthesis protein TAR RNA-binding protein 2 (TRBP). The use of enoxacin in human cell cultures and xenografted, orthotopic, and metastatic mouse models reveals a TRBP-dependent and cancer-specific growth-inhibitory effect of the drug. These results highlight the key role of disrupted miRNA expression patterns in tumorigenesis, and suggest a unique strategy for restoring the distorted microRNAome of cancer cells to a more physiological setting.

Do Alloreactivity and Prolonged Cold Ischemia Cause Different Elementary Lesions in Chronic Allograft Nephropathy

This study assesses the individual contributions of the nonalloreactive factor, cold ischemia (CI), and alloreactivity to late functional and structural renal graft changes, and examines the effect of the association of both factors on the progression of chronic allograft nephropathy. Lewis rats acted as receptors of kidneys from either Lewis or Fischer rats. For CI, kidneys were preserved for 5 hours. The rats were divided into four groups: Syn, syngeneic graft; SynI, syngeneic graft and CI; Allo, allogeneic graft; AlloI, allogeneic graft and CI. Renal function was assessed every 4 weeks for 24 weeks. Grafts were evaluated for acute inflammatory response at 1 week and for chronic histological damage at 24 weeks. Only when CI and allogenicity were combined did immediate posttransplant mortality occur, while survivors showed accelerated renal insufficiency that induced further mortality at 12 weeks after transplant. Solely ischemic rats developed renal insufficiency. Renal structural damage in ischemic rats was clearly tubulointerstitial, while significant vasculopathy and glomerulosclerosis appeared only in the allogeneic groups. There was increased infiltration of macrophages and expression of mRNA-transforming growth factor-beta1 in the ischemic groups, irrespective of the allogeneic background. The joint association of CI plus allogenicity significantly increased cellular infiltration at both early and late stages, aggravating tubulointerstitial and vascular damage considerably. In summary, CI is mainly responsible for tubulointerstitial damage, whereas allogenicity leads to vascular lesion. The association of both factors accelerates and aggravates the progression of experimental chronic allograft nephropathy.

BRCA1 epigenetic inactivation predicts sensitivity to platinum-based chemotherapy in breast and ovarian cancer.

Germline mutations in the BRCA1 or BRCA2 genes are associated with an increased risk of breast and ovarian cancer development. Both genes are involved in DNA repair, and tumors harboring genetic defects in them are thought to be more sensitive to DNA-damaging agents used in chemotherapy. However, as only a minority of breast and ovarian cancer patients carry BRCA1 or BRCA2 mutations, few patients are likely to benefit from these pharmacogenetic biomarkers. Herein, we show that, in cancer cell lines and xenografted tumors, BRCA1 CpG island promoter hypermethylation-associated silencing also predicts enhanced sensitivity to platinum-derived drugs to the same extent as BRCA1 mutations. Most importantly, BRCA1 hypermethylation proves to be a predictor of longer time to relapse and improved overall survival in ovarian cancer patients undergoing chemotherapy with cisplatin.

Germline Mutations in FAN1 Cause Hereditary Colorectal Cancer by Impairing DNA Repair

Identification of genes associated with hereditary cancers facilitates management of patients with family histories of cancer. We performed exome sequencing of DNA from 3 individuals from a family with colorectal cancer who met the Amsterdam criteria for risk of hereditary nonpolyposis colorectal cancer. These individuals had mismatch repair-proficient tumors and each carried nonsense variant in the FANCD2/FANCI-associated nuclease 1 gene (FAN1), which encodes a nuclease involved in DNA inter-strand cross-link repair. We sequenced FAN1 in 176 additional families with histories of colorectal cancer and performed in vitro functional analyses of the mutant forms of FAN1 identified. We detected FAN1 mutations in approximately 3% of families who met the Amsterdam criteria and had mismatch repair-proficient cancers with no previously associated mutations. These findings link colorectal cancer predisposition to the Fanconi anemia DNA repair pathway, supporting the connection between genome integrity and cancer risk.

Sunitinib Inhibits Tumor Growth and Synergizes with Cisplatin in Orthotopic Models of Cisplatin-Sensitive and Cisplatin-Resistant Human Testicular Germ Cell Tumors

Purpose: Germ cell tumors (GCT) of the testis are highly curable, but those patients who are refractory to cisplatin (CDDP)-based combination chemotherapy have a poor prognosis. Therefore, identifying new alternatives for treatment remains a priority. Several studies support an important role for angiogenesis in GCTs, suggesting that antiangiogenic treatment might be a good alternative. Sunitinib is an oral multitarget tyrosine kinase receptor inhibitor with antiangiogenic and antitumor activities. In the present study, we evaluated the effect of sunitinib, CDDP, or the combination of both drugs using an orthotopic model of human testicular GCT. Experimental Design: Mice were implanted with four different testicular tumors: a yolk sac, two choriocarcinomas, and a CDDP-resistant choriocarcinoma variant induced in mice by continuous exposure to CDDP. Mice were treated with vehicle, CDDP, sunitinib, or the combination of both drugs and their effects on tumors were analyzed. Results: We observed a significant inhibition in tumor growth accompanied by longer survival after sunitinib treatment. Combination therapy with CDDP significantly enhanced these effects. Sunitinib induced apoptosis, reduced tumor cell proliferation and tumor vasculature, and inhibited vascular endothelial growth factor receptor 1, 2, and 3 and platelet-derived growth factor receptor α phosphorylation without affecting phosphorylation of other tyrosine kinase receptors. More importantly, tumor growth inhibition induced by sunitinib was also observed in the induced CDDP-resistant choriocarcinoma model. Conclusions: Taken together, these results suggest that sunitinib might be a new alternative for treatment of CDDP-refractory patients.

Combined inhibition of DDR1 and Notch signaling is a therapeutic strategy for KRAS-driven lung adenocarcinoma

Patients with advanced Kirsten rat sarcoma viral oncogene homolog (KRAS)-mutant lung adenocarcinoma are currently treated with standard chemotherapy because of a lack of efficacious targeted therapies. We reasoned that the identification of mediators of Kras signaling in early mouse lung hyperplasias might bypass the difficulties that are imposed by intratumor heterogeneity in advanced tumors, and that it might unveil relevant therapeutic targets. Transcriptional profiling of KrasG12V-driven mouse hyperplasias revealed intertumor diversity with a subset that exhibited an aggressive transcriptional profile analogous to that of advanced human adenocarcinomas. The top-scoring gene in this profile encodes the tyrosine kinase receptor DDR1. The genetic and pharmacological inhibition of DDR1 blocked tumor initiation and tumor progression, respectively. The concomitant inhibition of both DDR1 and Notch signaling induced the regression of KRAS;TP53-mutant patient-derived lung xenografts (PDX) with a therapeutic efficacy that was at least comparable to that of standard chemotherapy. Our data indicate that the combined inhibition of DDR1 and Notch signaling could be an effective targeted therapy for patients with KRAS-mutant lung adenocarcinoma.

Epigenetic loss of the PIWI/piRNA machinery in human testicular tumorigenesis

Although most cancer research has focused in mRNA, non-coding RNAs are also an essential player in tumorigenesis. In addition to the well-recognized microRNAs, recent studies have also shown that epigenetic silencing by CpG island hypermethylation of other classes of non-coding RNAs, such as transcribed ultraconserved regions (T-UCRs) or small nucleolar RNAs (snoRNAs), also occur in human neoplasia. Herein we have studied the putative existence of epigenetic aberrations in the activity of PIWI proteins, an Argonaute family protein subclass, and the small regulatory PIWI-interacting RNAs (piRNAs) in testicular cancer, as the PIWI/piRNA pathway plays a critical role in male germline development. We have observed the existence of promoter CpG island hypermethylation-associated silencing of PIWIL1, PIWIL2, PIWIL4, and TDRD1 in primary seminoma and non-seminoma testicular tumors, in addition to testicular germ cell tumor cell lines. Most importantly, these epigenetic lesions occur in a context of piRNA downregulation and loss of DNA methylation of the LINE-1 repetitive sequences, one of the target genomic loci where the PIWI/piRNA machinery acts as a caretaker in non-transformed cells.

Rapamycin has dual opposing effects on proteinuric experimental nephropathies: is it a matter of podocyte damage?

BACKGROUND In clinical renal transplantation, an increase in proteinuria after conversion from calcineurin inhibitors to rapamycin has been reported. In contrast, there are studies showing a nephro-protective effect of rapamycin in proteinuric diseases characterized by progressive interstitial inflammatory fibrosis. METHODS Because of the contradictory reports concerning rapamycin on proteinuria, we examined proteinuria and podocyte damage markers on two renal disease models, with clearly different pathophysiological mechanisms: a glomerular toxico-immunological model induced by puromycin aminonucleoside, and a chronic hyperfiltration and inflammatory model by mass reduction, both treated with a fixed high rapamycin dose. RESULTS In puromycin groups, rapamycin provoked significant increases in proteinuria, together with a significant fall in podocin immunofluorescence, as well as clear additional damage to podocyte foot processes. Conversely, after mass reduction, rapamycin produced lower levels of proteinuria and amelioration of inflammatory and pro-fibrotic damage. In contrast to the puromycin model, higher glomerular podocin and nephrin expression and amelioration of glomerular ultrastructural damage were found. CONCLUSIONS We conclude that rapamycin has dual opposing effects on subjacent renal lesion, with proteinuria and podocyte damage aggravation in the glomerular model and a nephro-protective effect in the chronic inflammatory tubulointerstitial model. Rapamycin produces slight alterations in podocyte structure when acting on healthy podocytes, but it clearly worsens those podocytes damaged by other concomitant injury.