Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer.

Cancer-associated fibroblasts (CAFs) are the major components of the tumor microenvironment. They may drive tumor progression, although the mechanisms involved are still poorly understood. Exosomes have emerged as important mediators of intercellular communication in cancer. They mediate horizontal transfer of microRNAs (miRs), mRNAs and proteins, thus affecting breast cancer progression. Differential expression profile analysis identified three miRs (miRs -21, -378e, and -143) increased in exosomes from CAFs as compared from normal fibroblasts. Immunofluorescence indicated that exosomes may be transferred from CAFs to breast cancer cells, releasing their cargo miRs. Breast cancer cells (BT549, MDA-MB-231, and T47D lines) exposed to CAF exosomes or transfected with those miRs exhibited a significant increased capacity to form mammospheres, increased stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent cell growth. These effects were reverted by transfection with anti-miRs. Similarly to CAF exosomes, normal fibroblast exosomes transfected with miRs -21, -378e, and -143 promoted the stemness and EMT phenotype of breast cancer cells. Thus, we provided evidence for the first time of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines. Indeed, CAFs strongly promote the development of an aggressive breast cancer cell phenotype.

MiR-24 induces chemotherapy resistance and hypoxic advantage in breast cancer.

Breast cancer remains one of the leading causes of cancer mortality among women. It has been proved that the onset of cancer depends on a very small pool of tumor cells with a phenotype similar to that of normal adult stem cells. Cancer stem cells (CSC) possess self-renewal and multilineage differentiation potential as well as a robust ability to sustain tumorigenesis. Evidence suggests that CSCs contribute to chemotherapy resistance and to survival under hypoxic conditions. Interestingly, hypoxia in turn regulates self-renewal in CSCs and these effects may be primarily mediated by hypoxic inducible factors (HIFs). Recently, microRNAs (miRNAs) have emerged as critical players in the maintenance of pluripotency and self-renewal in normal and cancer stem cells. Here, we demonstrate that miR-24 is upregulated in breast CSCs and that its overexpression increases the number of mammospheres and the expression of stem cell markers. MiR-24 also induces apoptosis resistance through the regulation of BimL expression. Moreover, we identify a new miR-24 target, FIH1, which promotes HIFα degradation: miR-24 increases under hypoxic conditions, causing downregulation of FIH1 and upregulation of HIF1α. In conclusion, miR-24 hampers chemotherapy-induced apoptosis in breast CSCs and increases cell resistance to hypoxic conditions through an FIH1-HIFα pathway.

RYK promotes the stemness of glioblastoma cells via the WNT/ ß-catenin pathway.

Glioblastoma multiforme (GBM) is characterized by a strong self-renewal potential and a poor differentiation state. Since receptor-like tyrosine kinase (RYK) activates the WNT/ß-catenin pathway essential for cancer stem cell maintenance, we evaluated its contribution in conferring stemness to GBM cells. Here, we report that Ryk (related-to-receptor tyrosine kinase), an atypical tyrosine kinase receptor, is upregulated in samples from GBM patients as well as in GSCs. Ryk overexpression confers stemness properties to GBM cells through the modulation of the canonical Wnt signaling and by promoting the activation of pluripotency-related transcription factor circuitry and neurosphere formation ability. In contrast, siRNA-mediated knockdown of Ryk expression suppresses this stem-like phenotype. Rescue experiments reveal that stemness-promoting activity of Ryk is attributable, at least in part, to ß-catenin stabilization. Furthermore, Ryk overexpression improves cell motility and anchorage independent cell growth. Taken together, our findings demonstrate that Ryk promotes stem cell-like and tumorigenic features to glioma cells its essential for the maintenance of GSCs and could be a target of novel therapies.

Aptamer-miRNA-212 Conjugate Sensitizes NSCLC Cells to TRAIL.

TNF-related apoptosis-inducing ligand (TRAIL) is a promising antitumor agent for its remarkable ability to selectively induce apoptosis in cancer cells, without affecting the viability of healthy bystander cells. The TRAIL tumor suppressor pathway is deregulated in many human malignancies including lung cancer. In human non-small cell lung cancer (NSCLC) cells, sensitization to TRAIL therapy can be restored by increasing the expression levels of the tumor suppressor microRNA-212 (miR-212) leading to inhibition of the anti-apoptotic protein PED/PEA-15 implicated in treatment resistance. In this study, we exploited a previously described RNA aptamer inhibitor of the tyrosine kinase receptor Axl (GL21.T) expressed on lung cancer cells, as a means to deliver miR-212 into human NSCLC cells expressing Axl. We demonstrate efficient delivery of miR-212 following conjugation of the miR to GL21.T (GL21.T-miR212 chimera). We show that the chimera downregulates PED and restores TRAIL-mediate cytotoxicity in cancer cells. Importantly, treatment of Axl+ lung cancer cells with the chimera resulted in (i) an increase in caspase activation and (ii) a reduction of cell viability in combination with TRAIL therapy. In conclusion, we demonstrate that the GL21.T-miR212 chimera can be employed as an adjuvant to TRAIL therapy for the treatment of lung cancer.

RenalGuard System for the prevention of acute kidney injury in patients undergoing transcatheter aortic valve implantation.

AIMS: We aimed to assess whether the RenalGuard™ System is effective in preventing acute kidney injury (AKI) following transcatheter aortic valve implantation (TAVI). METHODS AND RESULTS: Forty-eight consecutive patients with chronic kidney disease (CKD) scheduled for TAVI were assigned to: 1) hydration with sodium bicarbonate solution (Control group), or 2) hydration with RenalGuard Therapy (RenalGuard group). Hypotension was defined as periprocedural mean blood pressure <55 mmHg. The primary endpoint was the occurrence of AKI (i.e., an increase of ≥0.3 mg/dL in the serum creatinine concentration at seven days). AKI occurred in 10/26 (38.5%) patients in the Control group and in 1/22 (4.5%) patients in the RenalGuard group (p=0.005, odds ratio [OR] 0.076, 95% confidence interval [CI]: 0.009-0.66). RenalGuard Therapy protected against AKI (OR 0.71, 95% CI: 0.07-0.775, p=0.026), whereas post-procedural hypotension (OR 3.88, 95% CI: 1.06-14.24, p=0.040), and contrast media volume (OR 3.65, 95% CI: 1.15-5.75, p=0.043) increased the risk of AKI. CONCLUSIONS: This non-randomised pilot study suggests that RenalGuard Therapy may be effective in preventing AKI in CKD patients undergoing TAVI.

RenalGuard system in high-risk patients for contrast-induced acute kidney injury.

BACKGROUND: High urine flow rate (UFR) has been suggested as a target for effective prevention of contrast-induced acute kidney injury (CI-AKI). The RenalGuard therapy (saline infusion plus furosemide controlled by the RenalGuard system) facilitates the achievement of this target. METHODS: Four hundred consecutive patients with an estimated glomerular filtration rate ≤30 mL/min per 1.73 m(2) and/or a high predicted risk (according to the Mehran score ≥11 and/or the Gurm score >7%) treated by the RenalGuard therapy were analyzed. The primary end points were (1) the relationship between CI-AKI and UFR during preprocedural, intraprocedural, and postprocedural phases of the RenalGuard therapy and (2) the rate of acute pulmonary edema and impairment in electrolytes balance. RESULTS: Urine flow rate was significantly lower in the patients with CI-AKI in the preprocedural phase (208 ± 117 vs 283 ± 160 mL/h, P < .001) and in the intraprocedural phase (389 ± 198 vs 483 ± 225 mL/h, P = .009). The best threshold for CI-AKI prevention was a mean intraprocedural phase UFR ≥450 mL/h (area under curve 0.62, P = .009, sensitivity 80%, specificity 46%). Performance of percutaneous coronary intervention (hazard ratio [HR] 4.13, 95% CI 1.81-9.10, P < .001), the intraprocedural phase UFR <450 mL/h (HR 2.27, 95% CI 1.05-2.01, P = .012), and total furosemide dose >0.32 mg/kg (HR 5.03, 95% CI 2.33-10.87, P < .001) were independent predictors of CI-AKI. Pulmonary edema occurred in 4 patients (1%). Potassium replacement was required in 16 patients (4%). No patients developed severe hypomagnesemia, hyponatremia, or hypernatremia. CONCLUSIONS: RenalGuard therapy is safe and effective in reaching high UFR. Mean intraprocedural UFR ≥450 mL/h should be the target for optimal CI-AKI prevention.

miR-340 predicts glioblastoma survival and modulates key cancer hallmarks through down-regulation of NRAS.

Glioblastoma is the most common primary brain tumor in adults; with a survival rate of 12 months from diagnosis. However, a small subgroup of patients, termed long-term survivors (LTS), has a survival rate longer then 12-14 months. There is thus increasing interest in the identification of molecular signatures predicting glioblastoma prognosis and in how to improve the therapeutic approach. Here, we report miR-340 as prognostic tumor-suppressor microRNA for glioblastoma. We analyzed microRNA expression in > 500 glioblastoma patients and found that although miR-340 is strongly down-regulated in glioblastoma overall, it is up-regulated in LTS patients compared to short-term survivors (STS). Indeed, miR-340 expression predicted better prognosis in glioblastoma patients. Coherently, overexpression of miR-340 in glioblastoma cells was found to produce a tumor-suppressive activity. We identified NRAS mRNA as a critical, direct target of miR-340: in fact, miR-340 negatively influenced multiple aspects of glioblastoma tumorigenesis by down-regulating NRAS and downstream AKT and ERK pathways. Thus, we demonstrate that expression of miR-340 in glioblastoma is responsible for a strong tumor-suppressive effect in LTS patients by down-regulating NRAS. miR-340 may thus represent a novel marker for glioblastoma diagnosis and prognosis, and may be developed into a tool to improve treatment of glioblastoma.

MiR-221 promotes stemness of breast cancer cells by targeting DNMT3b.

Cancer stem cells (CSCs) are a small part of the heterogeneous tumor cell population possessing self-renewal and multilineage differentiation potential as well as a great ability to sustain tumorigenesis. The molecular pathways underlying CSC phenotype are not yet well characterized. MicroRNAs (miRs) are small noncoding RNAs that play a powerful role in biological processes. Early studies have linked miRs to the control of self-renewal and differentiation in normal and cancer stem cells. We aimed to study the functional role of miRs in human breast cancer stem cells (BCSCs), also named mammospheres. We found that miR-221 was upregulated in BCSCs compared to their differentiated counterpart. Similarly, mammospheres from T47D cells had an increased level of miR-221 compared to differentiated cells. Transfection of miR-221 in T47D cells increased the number of mammospheres and the expression of stem cell markers. Among miR-221's targets, we identified DNMT3b. Furthermore, in BCSCs we found that DNMT3b repressed the expression of various stemness genes, such as Nanog and Oct 3/4, acting on the methylation of their promoters, partially reverting the effect of miR-221 on stemness. We hypothesize that miR-221 contributes to breast cancer tumorigenicity by regulating stemness, at least in part through the control of DNMT3b expression.

Neutrophil Gelatinase-Associated Lipocalin and Contrast-Induced Acute Kidney Injury.

BACKGROUND: Neutrophil gelatinase-associated lipocalin (NGAL) is an early marker of acute kidney injury (AKI). METHODS AND RESULTS: Urine NGAL and serum NGAL (sNGAL) were assessed at 2, 6, 24, and 48 hours after contrast media (CM) exposure in 458 high-risk patients (development set). Optimal thresholds in predicting contrast-induced AKI (serum creatinine [sCr] increase ≥0.3 mg/dL at 48 hours after CM administration) were identified. Major adverse events (MAE; death, dialysis, nonfatal myocardial infarction, sustained kidney injury, and myocardial revascularization) at 1 year were assessed. In the development set, optimal thresholds for contrast-induced AKI occurred at 6 hours for both urine NGAL (≥20 ng/mL; 97% negative predictive value and 27% positive predictive value) and sNGAL (≥179 ng/mL; 93% negative predictive value and 20% positive predictive value). Furthermore, sNGAL ≥179 ng/mL at 6 hours was an independent predictor of 1-year MAE. 1-year MAE occurred in 27/198 patients (13.5%) with sNGAL <179 ng/mL and sCr <0.3 mg/dL, in 57/193 (29.5%) patients with only sNGAL ≥179 ng/mL, and in 37/67 (55%) patients with sCr ≥0.3 mg/dL. In additional 253 patients (validation set), no patient with urine NGAL <20 ng/mL or sNGAL <179 ng/mL at 6 hours developed contrast-induced AKI. Furthermore, 6/68 (9%) patients with sNGAL <179 ng/mL and sCr increase <0.3 mg/dL had 1-year MAE versus 17/57 (30%) patients with sNGAL ≥179 ng/mL and sCr increase <0.3 mg/dL and 8/16 (50%) patients with sCr increase ≥0.3 mg/dL. CONCLUSIONS: Urine NGAL <20 ng/mL and sNGAL <179 ng/mL at 6 hours are reliable markers for ruling out contrast-induced AKI. sNGAL ≥179 ng/mL at 6 hours predicts 1-year MAE. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01098032.

Contrast-induced acute kidney injury: potential new strategies.

PURPOSE OF REVIEW: Contrast-induced acute kidney injury (CI-AKI) is an impairment of renal function following contrast media administration in the absence of an alternative cause. It represents a powerful predictor of poor early and late outcomes. Here, we review the major strategies to prevent CI-AKI. RECENT FINDINGS: Hydration represents the gold standard as a prophylactic measure to prevent CI-AKI, acting by increasing urine flow rate and, thereby, by limiting the time of contact between the contrast media and the tubular epithelial cells. An optimal hydration regimen should be defined according to predefined clinical markers, such as urine flow rate, or left ventricular end-diastolic pressure. Recently, high-dose statins pretreatment has been included in the guidelines of CI-AKI prevention. However, uncertainty still exists on the efficacy of several compounds tested in both observational trials and randomized studies to prevent CI-AKI. Compounds evaluated include diuretics (furosemide), antioxidants (i.e. N-acetylcysteine and statins) and vasodilators (i.e. calcium antagonists, dopamine and fenoldopam). SUMMARY: Hydration still represents the most reliable strategy to prevent CI-AKI. New prophylactic strategies for acute kidney injury are still under investigation.

Novel biomarkers for contrast-induced acute kidney injury.

Biomarkers of acute kidney injury (AKI) may be classified in 2 groups: (1) those representing changes in renal function (e.g., serum creatinine or cystatin C and urine flow rate) and (2) those reflecting kidney damage (e.g., kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), interleukin-18, etc.). According to these 2 fundamental criteria, 4 subgroups have been proposed: (1) no marker change; (2) damage alone; (3) functional change alone; and (4) combined damage and functional change. Therefore, a new category of patients with "subclinical AKI" (that is, an increase in damage markers alone without simultaneous loss of kidney function) has been identified. This condition has been associated with higher risk of adverse outcomes (including renal replacement therapy and mortality) at followup. The ability to measure these physiological variables may lead to identification of patients at risk for AKI and early diagnosis of AKI and may lead to variables, which may inform therapeutic decisions.

MiR-221/222 target the DNA methyltransferase MGMT in glioma cells.

Glioblastoma multiforme (GBM) is one of the most deadly types of cancer. To date, the best clinical approach for treatment is based on administration of temozolomide (TMZ) in combination with radiotherapy. Much evidence suggests that the intracellular level of the alkylating enzyme O(6)-methylguanine-DNA methyltransferase (MGMT) impacts response to TMZ in GBM patients. MGMT expression is regulated by the methylation of its promoter. However, evidence indicates that this is not the only regulatory mechanism present. Here, we describe a hitherto unknown microRNA-mediated mechanism of MGMT expression regulation. We show that miR-221 and miR-222 are upregulated in GMB patients and that these paralogues target MGMT mRNA, inducing greater TMZ-mediated cell death. However, miR-221/miR-222 also increase DNA damage and, thus, chromosomal rearrangements. Indeed, miR-221 overexpression in glioma cells led to an increase in markers of DNA damage, an effect rescued by re-expression of MGMT. Thus, chronic miR-221/222-mediated MGMT downregulation may render cells unable to repair genetic damage. This, associated also to miR-221/222 oncogenic potential, may poor GBM prognosis.

Therapeutic strategies to prevent contrast-induced acute kidney injury.

PURPOSE OF REVIEW: Contrast-induced acute kidney injury (CI-AKI) accounts for approximately 10% of all causes of hospital-acquired renal failure, causes a prolonged in-hospital stay, and represents a powerful predictor of poor early and late outcome. Here, we highlight endpoints used to assess major strategies to prevent CI-AKI. RECENT FINDINGS: A general consensus exists on the beneficial prophylactic effect of hydration. This seems to act by increasing urine flow rate and, thereby, by limiting the time of contact between the contrast media and the epithelial tubular cells. On the contrary, both observational trials and randomized studies are often controversial in their conclusions on the efficacy of several drugs tested to prevent CI-AKI. Compounds evaluated include diuretics (furosemide), antioxidants (i.e., N-acetylcysteine and statins), and vasodilators (i.e., calcium antagonists, dopamine, and fenoldopam). Due to the negative and/or controversial clinical results, none of these drugs has been currently recommended to prevent CI-AKI. CONCLUSION: More reliable markers of acute kidney injury and new prophylactic strategies are warranted to prevent the incidence of CI-AKI.

Impact of a high loading dose of atorvastatin on contrast-induced acute kidney injury.

BACKGROUND: The role of statins in the prevention of contrast-induced acute kidney injury (CIAKI) is controversial. METHODS AND RESULTS: First, we investigated the in vivo effects of atorvastatin on CIAKI. Patients with chronic kidney disease enrolled in the Novel Approaches for Preventing or Limiting Events (NAPLES) II trial were randomly assigned to (1) the atorvastatin group (80 mg within 24 hours before contrast media [CM] exposure; n=202) or (2) the control group (n=208). All patients received a high dose of N-acetylcysteine and sodium bicarbonate solution. Second, we investigated the in vitro effects of atorvastatin pretreatment on CM-mediated modifications of intracellular pathways leading to apoptosis or survival in renal tubular cells. CIAKI (ie, an increase >10% of serum cystatin C concentration within 24 hours after CM exposure) occurred in 9 of 202 patients in the atorvastatin group (4.5%) and in 37 of 208 patients in the control group (17.8%) (P=0.005; odds ratio=0.22; 95% confidence interval, 0.07-0.69). CIAKI rate was lower in the atorvastatin group in both diabetics and nondiabetics and in patients with moderate chronic kidney disease (estimated glomerular filtration rate, 31-60 mL/min per 1.73 m(2)). In the in vitro model, pretreatment with atorvastatin (1) prevented CM-induced renal cell apoptosis by reducing stress kinases activation and (2) restored the survival signals (mediated by Akt and ERK pathways). CONCLUSIONS: A single high loading dose of atorvastatin administered within 24 hours before CM exposure is effective in reducing the rate of CIAKI. This beneficial effect is observed only in patients at low to medium risk.