MicroRNA-34A inhibits the growth,invasion and metastasis of gastric cancer by targeting PDGFR and MET expression

Within the family of RTKs (receptor tyrosine kinases), PDGFR (platelet-derived growth factor receptor) has been implicated in carcinogenesis and tumour development. miRNAs (microRNAs), which can target the mRNAs (messenger RNAs) of cancer-associated genes, are abnormally expressed in various cancers. In this study, our aim was to identify the miRNAs that target PDGFR-α/β and to study the functions of these miRNAs. miR-34a was predicted to target PDGFR, and luciferase reporter assays showed that miR-34a could directly target PDGFR. Meanwhile, we found that miR-34a was down-regulated in gastric cancer tissues and was associated with metastasis. Our findings showed that miR-34a could inhibit gastric cancer cell migration, invasion and proliferation, but these tumourigenic properties were only partially restored when PDGFR-α/β was overexpressed. In subsequent experiments, we found that the overexpression of both PDGFR and MET could completely restore the gastric cancer tumourigenic properties. Moreover, the cancer-associated cell signalling pathway was studied, and we found that miR-34a could inhibit Akt [PKB (protein kinase B)] phosphorylation, which was restored by the overexpression of both PDGFR and MET. In conclusion, miR-34a may act as a potential tumour suppressor in gastric cancer and is associated with the mechanisms of gastric cancer metastasis; miR-34a can inhibit gastric cancer tumourigenesis by targeting PDGFR and MET through the PI3K (phosphoinositide 3-kinase)/Akt pathway.     

Tocopherol Transfer Protein Sensitizes Prostate Cancer Cells to Vitamin E

Prostate cancer is a major cause of mortality in men in developed countries. It has been reported that the naturally occurring antioxidant α-tocopherol (vitamin E) attenuates prostate cancer cell proliferation in cultured cells and mouse models. We hypothesized that overexpression of the tocopherol transfer protein (TTP), a vitamin E-binding protein that regulates tocopherol status, will sensitize prostate cancer cells to the anti-proliferative actions of the vitamin. To test this notion, we manipulated the expression levels of TTP in cultured prostate cells (LNCaP, PC3, DU145, and RWPE-1) using overexpression and knockdown approaches. Treatment of cells with tocopherol caused a time- and dose-dependent inhibition of cell proliferation. Overexpression of TTP dramatically sensitized the cells to the apoptotic effects of α-tocopherol, whereas reduction (“knockdown”) of TTP expression resulted in resistance to the vitamin. TTP levels also augmented the inhibitory effects of vitamin E on proliferation in semi-solid medium. The sensitizing effects of TTP were paralleled by changes in the intracellular accumulation of a fluorescent analog of vitamin E and by a reduction in intracellular levels of reactive oxygen species and were not observed when a naturally occurring, ligand binding-defective mutant of TTP was used. We conclude that TTP sensitizes prostate cancer cells to the anti-proliferative effects of vitamin E and that this activity stems from the ability of protein to increase the intracellular accumulation of the antioxidant. These observations support the notion that individual changes in the expression level or activity of TTP may determine the responsiveness of prostate cancer patients to intervention strategies that utilize vitamin E.     

MiR-34a/c-Dependent PDGFR-α/β Downregulation Inhibits Tumorigenesis and Enhances TRAIL-Induced Apoptosis in Lung Cancer

Lung cancer is the leading cause of cancer mortality in the world today. Although some advances in lung cancer therapy have been made, patient survival is still poor. MicroRNAs (miRNAs) can act as oncogenes or tumor-suppressor genes in human malignancy. The miR-34 family consists of tumor-suppressive miRNAs, and its reduced expression has been reported in various cancers, including non-small cell lung cancer (NSCLC). In this study, we found that miR-34a and miR-34c target platelet-derived growth factor receptor alpha and beta (PDGFR-α and PDGFR-β), cell surface tyrosine kinase receptors that induce proliferation, migration and invasion in cancer. MiR-34a and miR-34c were downregulated in lung tumors compared to normal tissues. Moreover, we identified an inverse correlation between PDGFR-α/β and miR-34a/c expression in lung tumor samples. Finally, miR-34a/c overexpression or downregulation of PDGFR-α/β by siRNAs, strongly augmented the response to TNF-related apoptosis inducing ligand (TRAIL) while reducing migratory and invasive capacity of NSCLC cells.     

The Potential Regulatory Mechanisms of miR-196a in Huntington’s Disease through Bioinformatic Analyses

High throughput screening is a powerful tool to identify the potential candidate molecules involved during disease progression. However, analysis of complicated data is one of the most challenging steps on the way to obtaining useful results from this approach. Previously, we showed that a specific miRNA, miR-196a, could ameliorate the pathological phenotypes of Huntington’s disease (HD) in different models, and performed high throughput screening by using the striatum of transgenic mice. In this study, we further tried to identify the potential regulatory mechanisms using different bioinformatic tools, including Database for Annotation, Visualization and Integrated Discovery (DAVID), Molecular Signatures Database (MSigDB), TargetScan and MetaCore. The results showed that miR-196a dominantly altered “ABC transporters”, “RIG-I-like receptor signaling pathway”, immune system”, “adaptive immune system”,“tissue remodeling and wound repair” and “cytoskeleton remodeling”. In addition, miR-196a also changed the expression of several well-defined pathways of HD, such as apoptosis and cell adhesion. Since these analyses showed the regulatory pathways are highly related to the modification of the cytoskeleton, we further confirmed that miR-196a could enhance the neurite outgrowth in neuroblastoma cells, suggesting miR-196a might provide beneficial functions through the alteration of cytoskeleton structures. Since impairment of the cytoskeleton has been reported in several neuronal diseases, this study will provide not only the potential working mechanisms of miR-196a but also insights for therapeutic strategies for use with different neuronal diseases.     

Regulation of Neuronal Cell Cycle and Apoptosis by MicroRNA 34a

The cell cycle of neurons remains suppressed to maintain the state of differentiation and aberrant cell cycle reentry results in loss of neurons, which is a feature in neurodegenerative disorders like Alzheimer''s disease (AD). Present studies revealed that the expression of microRNA 34a (miR-34a) needs to be optimal in neurons, as an aberrant increase or decrease in its expression causes apoptosis. miR-34a keeps the neuronal cell cycle under check by preventing the expression of cyclin D1 and promotes cell cycle arrest. Neurotoxic amyloid β_1–42 peptide (Aβ₄₂) treatment of cortical neurons suppressed miR-34a, resulting in unscheduled cell cycle reentry, which resulted in apoptosis. The repression of miR-34a was a result of degradation of TAp73, which was mediated by aberrant activation of the MEK extracellular signal-regulated kinase (ERK) pathway by Aβ₄₂. A significant decrease in miR-34a and TAp73 was observed in the cortex of a transgenic (Tg) mouse model of AD, which correlated well with cell cycle reentry observed in the neurons of these animals. Importantly, the overexpression of TAp73α and miR-34a reversed cell cycle-related neuronal apoptosis (CRNA). These studies provide novel insights into how modulation of neuronal cell cycle machinery may lead to neurodegeneration and may contribute to the understanding of disorders like AD.     

Epigenetic regulation of microRNA genes and the role of miR-34b in cell invasion and motility in human melanoma

Invasive melanoma is the most lethal form of skin cancer. The treatment of melanoma-derived cell lines with 5-aza-2'-deoxycytidine (5-Aza-dC) markedly increases the expression of several miRNAs, suggesting that the miRNA-encoding genes might be epigenetically regulated, either directly or indirectly, by DNA methylation. We have identified a group of epigenetically regulated miRNA genes in melanoma cells, and have confirmed that the upstream CpG island sequences of several such miRNA genes are hypermethylated in cell lines derived from different stages of melanoma, but not in melanocytes and keratinocytes. We used direct DNA bisulfite and immunoprecipitated DNA (Methyl-DIP) to identify changes in CpG island methylation in distinct melanoma patient samples classified as primary in situ, regional metastatic, and distant metastatic. Two melanoma cell lines (WM1552C and A375 derived from stage 3 and stage 4 human melanoma, respectively) were engineered to ectopically express one of the epigenetically modified miRNA: miR-34b. Expression of miR-34b reduced cell invasion and motility rates of both WM1552C and A375, suggesting that the enhanced cell invasiveness and motility observed in metastatic melanoma cells may be related to their reduced expression of miR-34b. Total RNA isolated from control or miR-34b-expressing WM1552C cells was subjected to deep sequencing to identify gene networks around miR-34b. We identified network modules that are potentially regulated by miR-34b, and which suggest a mechanism for the role of miR-34b in regulating normal cell motility and cytokinesis.     

MicroRNome Analysis Unravels the Molecular Basis of SARS Infection in Bronchoalveolar Stem Cells

Severe acute respiratory syndrome (SARS), caused by the coronavirus SARS-CoV, is an acute infectious disease with significant mortality. A typical clinical feature associated with SARS is pulmonary fibrosis and associated lung failure. In the aftermath of the SARS epidemic, although significant progress towards understanding the underlying molecular mechanism of the infection has been made, a large gap still remains in our knowledge regarding how SARS-CoV interacts with the host cell at the onset of infection. The rapidly changing viral genome adds another variable to this equation. We have focused on a novel concept of microRNA (miRNA)–mediated host–virus interactions in bronchoalveolar stem cells (BASCs) at the onset of infection by correlating the “BASC–microRNome” with their targets within BASCs and viral genome. This work encompasses miRNA array data analysis, target prediction, and miRNA–mRNA enrichment analysis and develops a complex interaction map among disease-related factors, miRNAs, and BASCs in SARS pathway, which will provide some clues for diagnostic markers to view an overall interplay leading to disease progression. Our observation reveals the BASCs (Sca-1+ CD34+ CD45- Pecam-), a subset of Oct-4+ ACE2+ epithelial colony cells at the broncho-alveolar duct junction, to be the prime target cells of SARS-CoV infection. Upregulated BASC miRNAs-17*, -574-5p, and -214 are co-opted by SARS-CoV to suppress its own replication and evade immune elimination until successful transmission takes place. Viral Nucleocapsid and Spike protein targets seem to co-opt downregulated miR-223 and miR-98 respectively within BASCs to control the various stages of BASC differentiation, activation of inflammatory chemokines, and downregulation of ACE2. All these effectively accounts for a successful viral transmission and replication within BASCs causing continued deterioration of lung tissues and apparent loss of capacity for lung repair. Overall, this investigation reveals another mode of exploitation of cellular miRNA machinery by virus to their own advantage.     

Oxidative DNA Damage in the Prostate May Predispose Men to a Higher Risk of Prostate Cancer

DNA damage has been associated with prostate cancer risk. Men who were referred for initial prostate biopsy for elevated prostate-specific antigen or abnormal digital rectal examination are often found with no cancer but have a higher risk of developing prostate cancer than the general population of men in their lifetime. In this study, we investigated whether DNA damage is one of the factors that predispose these men referred for prostate biopsies to a higher risk of prostate cancer. We found significantly elevated levels of 8-oxo-2-deoxyguanosine immunoreactivity in the prostates of the referred men (n = 50) in comparison to the control prostates of men (n = 32) with no indication for referral for prostate biopsy. Twelve of these control men were healthy middle-aged men and 20 of them were older men whose conditions were diagnosed with bladder cancer but with normal serum prostate-specific antigen and digital rectal examination and no evidence of prostate disease. In all the 8-oxo-2-deoxyguanosine-positive prostates, we detected phosphorylation of the ataxia telangiectasia mutated kinase and expression of the immune-stimulatory molecule MIC in the prostate epithelium. These data suggest that: 1) oxidative DNA damage has occurred in the “referred” but pathologically normal prostates, indicating that these prostates may be subjected to genomic instability and eventually neoplastic transformation; 2) in response to DNA damage, two surveillance pathways, represented by ataxia telangiectasia mutated phosphorylation and induction of the NKG2D ligand MIC, were activated to prevent tumorigenesis.     

Blood-Based Biomarkers of Aggressive Prostate Cancer

Purpose

Prostate cancer is a bimodal disease with aggressive and indolent forms. Current prostate-specific-antigen testing and digital rectal examination screening provide ambiguous results leading to both under-and over-treatment. Accurate, consistent diagnosis is crucial to risk-stratify patients and facilitate clinical decision making as to treatment versus active surveillance. Diagnosis is currently achieved by needle biopsy, a painful procedure. Thus, there is a clinical need for a minimally-invasive test to determine prostate cancer aggressiveness. A blood sample to predict Gleason score, which is known to reflect aggressiveness of the cancer, could serve as such a test.

Materials and Methods

Blood mRNA was isolated from North American and Malaysian prostate cancer patients/controls. Microarray analysis was conducted utilizing the Affymetrix U133 plus 2·0 platform. Expression profiles from 255 patients/controls generated 85 candidate biomarkers. Following quantitative real-time PCR (qRT-PCR) analysis, ten disease-associated biomarkers remained for paired statistical analysis and normalization.

Results

Microarray analysis was conducted to identify 85 genes differentially expressed between aggressive prostate cancer (Gleason score ≥8) and controls. Expression of these genes was qRT-PCR verified. Statistical analysis yielded a final seven-gene panel evaluated as six gene-ratio duplexes. This molecular signature predicted as aggressive (ie, Gleason score ≥8) 55% of G6 samples, 49% of G7(3+4), 79% of G7(4+3) and 83% of G8-10, while rejecting 98% of controls.

Conclusion

In this study, we have developed a novel, blood-based biomarker panel which can be used as the basis of a simple blood test to identify men with aggressive prostate cancer and thereby reduce the overdiagnosis and overtreatment that currently results from diagnosis using PSA alone. We discuss possible clinical uses of the panel to identify men more likely to benefit from biopsy and immediate therapy versus those more suited to an “active surveillance” strategy.     

The microRNA -23b/-27b Cluster Suppresses the Metastatic Phenotype of Castration-Resistant Prostate Cancer Cells

MicroRNAs (miRs) are small, endogenous, non-coding RNAs that regulate the stability and/or translation of complementary mRNA targets. MiRs have emerged not only as critical modulators of normal physiologic processes, but their deregulation may significantly impact prostate and other cancers. The expression of miR-23b and miR-27b, which are encoded by the same miR cluster (miR-23b/-27b), are downregulated in metastatic, castration-resistant tumors compared to primary prostate cancer and benign tissue; however, their possible role in prostate cancer progression is unknown. We found that ectopic expression of miR-23b/-27b in two independent castration-resistant prostate cancer cell lines resulted in suppression of invasion and migration, as well as reduced survival in soft agar (a measure of anoikis). However, there was no effect of miR-23b/-27b on cell proliferation suggesting that these miRs function as metastasis (but not growth) suppressors in prostate cancer. Conversely, inhibition of miR-23b/-27b in the less aggressive androgen-dependent LNCaP prostate cancer cell line resulted in enhanced invasion and migration also without affecting proliferation. Mechanistically, we found that introduction of miR-23b/-27b in metastatic, castration-resistant prostate cancer cell lines resulted in a significant attenuation of Rac1 activity without affecting total Rac1 levels and caused increased levels of the tumor suppressor E-cadherin. Inhibition of these miRs had the opposite effect in androgen-dependent LNCaP cells. These results suggest that miR-23b/-27b are metastasis suppressors that might serve as novel biomarkers and therapeutic agents for castration-resistant disease.     

The Axl-Regulating Tumor Suppressor miR-34a Is Increased in ccRCC but Does Not Correlate with Axl mRNA or Axl Protein Levels

Background

High expression of the receptor tyrosine kinase Axl is associated with poor prognosis in patients with Renal Cell Carcinoma (RCC), the most common malignancy of the kidney. The miR-34a has been shown to directly regulate Axl in cancer cells. The miR-34a is a mediator of p53-dependent tumor suppression, and low expression of miR-34a has been associated with worse prognosis in several cancers. Our aim was to elucidate whether miR-34a or the other members of the miR-34 family (miR-34b/c) regulate Axl in RCC.

Methodology and Results

Using western blot, flow cytometry, and RT-qPCR, we showed that Axl mRNA and protein are downregulated in 786-O cells by miR-34a and miR-34c but not by miR-34b. A luciferase reporter assay demonstrated direct interaction between the Axl 3’ UTR and miR-34a and miR-34c. The levels of miR-34a/b/c were measured in tumor tissue in a cohort of 198 RCC patients, and the levels of miR-34a were found to be upregulated in clear cell RCC (ccRCC) tumors, but not associated with patient outcome. Neither of the miR-34 family members correlated with Axl mRNA, soluble Axl protein in serum, nor with immunohistochemistry of Axl in tumor tissue. In addition, we measured mRNA levels of a known miR-34a target, HNF4A, and found the HNF4A levels to be decreased in ccRCC tumors, but unexpectedly correlated positively rather than negatively with miR-34a.

Conclusions

Although miR-34a and miR-34c can regulate Axl expression in vitro, our data indicates that the miR-34 family members are not the primary regulators of Axl expression in RCC.     

Circulating MicroRNAs as Easy-to-Measure Aging Biomarkers in Older Breast Cancer Patients: Correlation with Chronological Age but Not with Fitness/Frailty Status

Circulating microRNAs (miRNAs) hold great promise as easily accessible biomarkers for diverse (patho)physiological processes, including aging. We have compared miRNA expression profiles in cell-free blood from older versus young breast cancer patients, in order to identify “aging miRNAs” that can be used in the future to monitor the impact of chemotherapy on the patient’s biological age. First, we assessed 175 miRNAs that may possibly be present in serum/plasma in an exploratory screening in 10 young and 10 older patients. The top-15 ranking miRNAs showing differential expression between young and older subjects were further investigated in an independent cohort consisting of another 10 young and 20 older subjects. Plasma levels of miR-20a-3p, miR-30b-5p, miR106b, miR191 and miR-301a were confirmed to show significant age-related decreases (all p≤0.004). The remaining miRNAs included in the validation study (miR-21, miR-210, miR-320b, miR-378, miR-423-5p, let-7d, miR-140-5p, miR-200c, miR-374a, miR376a) all showed similar trends as observed in the exploratory screening but these differences did not reach statistical significance. Interestingly, the age-associated miRNAs did not show differential expression between fit/healthy and non-fit/frail subjects within the older breast cancer cohort of the validation study and thus merit further investigation as true aging markers that not merely reflect frailty.     

β-TrCP Inhibition Reduces Prostate Cancer Cell Growth via Upregulation of the Aryl Hydrocarbon Receptor

Background

Prostate cancer is a common and heterogeneous disease, where androgen receptor (AR) signaling plays a pivotal role in development and progression. The initial treatment for advanced prostate cancer is suppression of androgen signaling. Later on, essentially all patients develop an androgen independent stage which does not respond to anti hormonal treatment. Thus, alternative strategies targeting novel molecular mechanisms are required. β-TrCP is an E3 ligase that targets various substrates essential for many aspects of tumorigenesis.

Methodology/Principal Findings

Here we show that β-TrCP depletion suppresses prostate cancer and identify a relevant growth control mechanism. shRNA targeted against β-TrCP reduced prostate cancer cell growth and cooperated with androgen ablation in vitro and in vivo. We found that β-TrCP inhibition leads to upregulation of the aryl hydrocarbon receptor (AhR) mediating the therapeutic effect. This phenomenon could be ligand independent, as the AhR ligand 2,3,7,8-Tetrachlorodibenzo-p-Dioxin (TCDD) did not alter prostate cancer cell growth. We detected high AhR expression and activation in basal cells and atrophic epithelial cells of human cancer bearing prostates. AhR expression and activation is also significantly higher in tumor cells compared to benign glandular epithelium.

Conclusions/Significance

Together these observations suggest that AhR activation may be a cancer counteracting mechanism in the prostate. We maintain that combining β-TrCP inhibition with androgen ablation could benefit advanced prostate cancer patients.