MicroRNA regulation of CYP 1A2, CYP3A4 and CYP2E1 expression in acetaminophen toxicity.

MicroRNAs (miRNAs) that regulate the cytochrome P-450 isoforms involved in acetaminophen (APAP) toxicity were examined in HepaRG cells treated with APAP (20 mM). In-vitro studies found that APAP protein adducts were increased at 1 h, followed by ALT increases at 12 and 24 h. CYP1A2, CYP3A4 and CYP2E1 mRNA levels were decreased, while miRNAs were increased for miR-122-5p, miR-378a-5p, miR-27b-3p at 6 h and miR-125b-5p at 12 h and miR-27b-3p at 24 h. Putative miRNA binding sites on the 3'UTRs of the CYPs were identified in-silico. Overexpression of miR-122-5p and miR-378a-5p in cells suppressed protein expression of CYP1A2, CYP3A4 and CYP2E1. Luciferase reporter assays confirmed the interaction between miR-122 and the 3'UTR of the CYP1A2 and CYP3A4. Thus, the in-vitro experiments showed that miR-122-5p and miR-378a-5p upregulation were associated with translational repression of CYPs. Serum samples of children with APAP overdose had significant elevation of miR-122-5p, miR-378a-5p, miR-125b-5p and miR-27b-3p, compared to healthy controls and receiver operator curves of the miRNAs had AUCs of 91 to 100%. Collectively, the data suggest that miRNA elevations in APAP toxicity represent a regulatory response to modify CYP1A2, CYP3A4 and CYP2E1 translation due to cellular stress and injury.

Integrated Genomics Identifies miR-32/MCL-1 Pathway as a Critical Driver of Melanomagenesis: Implications for miR-Replacement and Combination Therapy.

AIMS: Cutaneous malignant melanoma is among the deadliest human cancers, broadly resistant to most clinical therapies. A majority of patients with BRAFV600E melanomas respond well to inhibitors such as vemurafenib, but all ultimately relapse. Moreover, there are no viable treatment options available for other non-BRAF melanoma subtypes in the clinic. A key to improving treatment options lies in a better understanding of mechanisms underlying melanoma progression, which are complex and heterogeneous. METHODS: In this study we integrated gene and microRNA (miRNA) expression data from genetically engineered mouse models of highly and poorly malignant melanocytic tumors, as well as available human melanoma databases, and discovered an important role for a pathway centered on a tumor suppressor miRNA, miR-32. RESULTS: Malignant tumors frequently exhibited poor expression of miR-32, whose targets include NRAS, PI3K and notably, MCL-1. Accordingly, MCL-1 was often highly expressed in melanomas, and when knocked down diminished oncogenic potential. Forced MCL-1 overexpression transformed immortalized primary mouse melanocytes, but only when also expressing activating mutations in BRAF, CRAF or PI3K. Importantly, both miR-32 replacement therapy and the MCL-1-specific antagonist sabutoclax demonstrated single-agent efficacy, and acted synergistically in combination with vemurafenib in preclinical melanoma models. CONCLUSIONS: We here identify miR-32/MCL-1 pathway members as key early genetic events driving melanoma progression, and suggest that their inhibition may be an effective anti-melanoma strategy irrespective of NRAS, BRAF, and PTEN status.

Keratinocyte Induced Differentiation of Mesenchymal Stem Cells into Dermal Myofibroblasts: A Role in Effective Wound Healing.

We have previously demonstrated that human mesenchymal stem cells (hMSCs) migrate toward human keratinocytes as well as toward conditioned medium from cultured human keratinocytes (KCM) indicating that the hMSCs respond to signals from keratinocytes [1]. Using fluorescently labeled cells we now show that in vitro hMSCs appear to surround keratinocytes, and this organization is recapitulated in vivo. Incubation of hMSCs with KCM induced dermal myofibroblast like differentiation characterized by expression of cytoskeletal markers and increased expression of cytokines including SDF-1, IL-8, IL-6 and CXCL5. Interaction of keratinocytes with hMSCs appears to be important in the wound healing process. Therapeutic efficacy of hMSCs in wound healing was examined in two animal models representing normal and chronic wound healing. Accelerated wound healing was observed when hMSCs and KCM exposed hMSCs (KCMSCs) were injected near wound site in nude and NOD/SCID mice. Long term follow up of wound healing revealed that in the hMSC treated wounds there was little evidence of residual scarring. These dermal myofibroblast like hMSCs add to the wound healing process. Together, the keratinocyte and hMSCs morphed dermal myofibroblast like cells as well as the factors secreted by these cells support wound healing with minimal scarring. The ability of hMSCs to support wound healing process represents another striking example of the importance of keratinocyte and hMSCs interplay in the wound microenvironment resulting in effective wound healing with minimal scarring.

Axl receptor tyrosine kinase is up-regulated in metformin resistant prostate cancer cells.

Recent epidemiological studies showed that metformin, a widely used anti-diabetic drug might prevent certain cancers. Metformin also has an anti-proliferative effect in preclinical studies of both hematologic malignancies as well as solid cancers and clinical studies testing metformin as an anti-cancer drug are in progress. However, all cancer types do not respond to metformin with the same effectiveness or acquire resistance. To understand the mechanism of acquired resistance and possibly its mechanism of action as an anti-proliferative agent, we developed metformin resistant LNCaP prostate cancer cells. Metformin resistant LNCaP cells had an increased proliferation rate, increased migration and invasion ability as compared to the parental cells, and expressed markers of epithelial-mesenchymal transition (EMT). A detailed gene expression microarray comparing the resistant cells to the wild type cells revealed that Edil2, Ereg, Axl, Anax2, CD44 and Anax3 were the top up-regulated genes and calbindin 2 and TPTE (transmembrane phosphatase with tensin homology) and IGF1R were down regulated. We focused on Axl, a receptor tyrosine kinase that has been shown to be up regulated in several drug resistance cancers. Here, we show that the metformin resistant cell line as well as castrate resistant cell lines that over express Axl were more resistant to metformin, as well as to taxotere compared to androgen sensitive LNCaP and CWR22 cells that do not overexpress Axl. Forced overexpression of Axl in LNCaP cells decreased metformin and taxotere sensitivity and knockdown of Axl in resistant cells increased sensitivity to these drugs. Inhibition of Axl activity by R428, a small molecule Axl kinase inhibitor, sensitized metformin resistant cells that overexpressed Axl to metformin. Inhibitors of Axl may enhance tumor responses to metformin and other chemotherapy in cancers that over express Axl.

Molecular profile of clonal strains of human skeletal stem/progenitor cells with different potencies.

Bone marrow stromal cells (BMSCs, also known as bone marrow-derived mesenchymal stem cells) are fibroblastic reticular cells, a subset of which is composed of multipotent skeletal stem cells (SSCs). SSCs/BMSCs are able to recreate a bone/marrow organ in vivo. To determine differences between clonogenic multipotent SSCs and similarly clonogenic but non-multipotent BMSCs, we established single colony-derived strains (SCDSs, initiated by individual Colony Forming Unit-Fibroblasts) and determined their differentiation capacity by vivo transplantation. In this series of human SCDSs (N=24), 20.8% formed fibrous tissue (F), 66.7% formed bone (B), and 12.5% formed a bone/marrow organ, and thus were multipotent (M). RNA isolated from 12 SCDSs just prior to transplantation was analyzed by microarray. Although highly similar, there was variability from one SCDS to another, and SCDSs did not strictly segregate into the three functional groups (F, B or M) by unsupervised hierarchical clustering. We then compared 3 F-SCDSs to 3 M-SCDSs that did segregate. Genes associated with skeletogenesis, osteoblastogeneis, hematopoiesis, and extracellular matrix were over-represented in M-SCDSs compared with F-SCDSs. These results highlight the heterogeneity of SSCs/BMSCs, even between functionally similar SCDSs, but also indicate that differences can be detected that may shed light on the character of the SSC.

Bone marrow skeletal stem/progenitor cell defects in dyskeratosis congenita and telomere biology disorders.

Dyskeratosis congenita (DC) is an inherited multisystem disorder, characterized by oral leukoplakia, nail dystrophy, and abnormal skin pigmentation, as well as high rates of bone marrow (BM) failure, solid tumors, and other medical problems such as osteopenia. DC and telomere biology disorders (collectively referred to as TBD here) are caused by germline mutations in telomere biology genes leading to very short telomeres and limited proliferative potential of hematopoietic stem cells. We found that skeletal stem cells (SSCs) within the BM stromal cell population (BMSCs, also known as BM-derived mesenchymal stem cells), may contribute to the hematologic phenotype. TBD-BMSCs exhibited reduced clonogenicity, spontaneous differentiation into adipocytes and fibrotic cells, and increased senescence in vitro. Upon in vivo transplantation into mice, TBD-BMSCs failed to form bone or support hematopoiesis, unlike normal BMSCs. TERC reduction (a TBD-associated gene) in normal BMSCs by small interfering TERC-RNA (siTERC-RNA) recapitulated the TBD-BMSC phenotype by reducing proliferation and secondary colony-forming efficiency, and by accelerating senescence in vitro. Microarray profiles of control and siTERC-BMSCs showed decreased hematopoietic factors at the messenger RNA level and decreased secretion of factors at the protein level. These findings are consistent with defects in SSCs/BMSCs contributing to BM failure in TBD.

MicroRNAs as promising biomarkers in cancer diagnostics.

Cumulating data suggest that small noncoding-RNAs such as microRNAs (miRNAs) can be utilized as potential biomarkers for the diagnosis and prognosis of a variety of diseases such as cancer, neurological disorders, cardiovascular disease and Type-II diabetes, etc. MiRNAs can be utilized not only for monitoring of treatments but also for patient stratifications. The Tenth Annual miRNA as Biomarkers and Diagnostics conference, 2014, organized in Boston, MA, was primarily focused on recent advancements in the field of miRNA in the early detection of disease, monitoring tumor growth/progression and its potential for precision medicine. This article summarizes findings presented in the miRNA biomarker as cancer diagnostics session. The overarching projections from this and other sessions were that miRNAs are now well established as regulators of tumorigenesis and can be utilized not only as potential biomarkers for the diagnosis and prognosis of a disease but also are useful in patient stratifications and treatment response.

Non-coding RNAs as clinical biomarkers for cancer diagnosis and prognosis.

Developing more precise diagnostics approaches to predict cancer progression and prognosis is the key to precision medicine. Overwhelming evidence now suggests that small non-coding RNAs such as miRNAs can be useful tools as biomarkers for molecular diagnostics. miRNAs can serve as biomarkers in a variety of diseases, such as neurological disorders, cardiovascular disease, Type II diabetes, cancer and so on. miRNAs can not only be utilized for monitoring treatment but also for patient stratification and hence are promising predictive biomarkers in cancer progression and prognosis, as well as in predicting drug response. This article focuses on some of the recent findings in the field of miRNA biomarkers and discusses its implications for cancer diagnostics and precision medicine.

Cell-free derivatives from mesenchymal stem cells are effective in wound therapy.

AIM: To compare the efficacy of cell-free derivatives from Bone marrow derived human mesenchymal stem cells (hMSCs) in wound therapy. METHODS: hMSCs have been shown to play an important role in wound therapy. The present study sought to compare efficacy of hMSCs and cell-free derivatives of hMSCs, which may be clinically more relevant as they are easier to prepare, formulate and transport. hMSCs were isolated from human bone marrow and cultured. Multi lineage differentiation of hMSCs was performed to confirm their identity. The ability of hMSCs to migrate was evaluated using in vitro and in vivo migration assays. Cell lysates and conditioned medium concentrate was prepared from hMSCs (see Methods for details). Wounds were induced in mice and wound areas were measure before and after cell and cell-free derivative treatment. RNA and proteins were extracted from the skin and cytokine levels were measured. RESULTS: Co-culture of hMSCs with keratinocytes resulted in increased expression of CXCL-12 (SDF1) and ENA78 (CXCL-5) in the conditioned media indicating that the hMSCs can respond to signals from keratinocytes. Accelerated wound closure was observed when hMSCs were injected near the site of excisional wounds in athymic as well as NOD/SCID mice. Interestingly, cell-free lysates prepared from hMSCs were also effective in inducing accelerated wound closure and increased expression of SDF1 and CXCL-5 at the wound bed. Additionally, concentrated media from hMSCs as well as an emulsion containing lysates prepared from hMSCs was also found to be more effective in rapid re-epithelialization than fibroblasts or vehicle-alone control. Use of cell-free derivatives may help replace expensive wound care approaches including use of growth factors, epidermal/dermal substitutes, synthetic membranes, cytokines, and matrix components, and most importantly avoid transmission of pathogens from human and animal products. CONCLUSION: These results encourage development of derivatives of hMSCs for wound care and re-epithelialization applications.

MiR-24 tumor suppressor activity is regulated independent of p53 and through a target site polymorphism.

MicroRNAs (miRNAs) are predicted to regulate approximately 30% of all human genes; however, only a few miRNAs have been assigned their targets and specific functions. Here we demonstrate that miR-24, a ubiquitously expressed miRNA, has an anti-proliferative effect independent of p53 function. Cell lines with differential p53 status were used as a model to study the effects of miR-24 on cell proliferation, cell cycle control, gene regulation and cellular transformation. Overexpression of miR-24 in six different cell lines, independent of p53 function, inhibited cell proliferation and resulted in G2/S cell cycle arrest. MiR-24 over expression in cells with wt-p53 upregulated TP53 and p21 protein; however, in p53-null cells miR-24 still induced cell cycle arrest without the involvement of p21. We show that miR-24 regulates p53-independent cellular proliferation by regulating an S-phase enzyme, dihydrofolate reductase (DHFR) a target of the chemotherapeutic drug methotrexate (MTX). Of interest, we found that a miR-24 target site polymorphism in DHFR 3' UTR that results in loss of miR-24-function and high DHFR levels in the cell imparts a growth advantage to immortalized cells and induces neoplastic transformation. Of clinical significance, we found that miR-24 is deregulated in human colorectal cancer tumors and a subset of tumors has reduced levels of miR-24. A novel function for miR-24 as a p53-independent cell cycle inhibitory miRNA is proposed.

Translational modulation of proteins expressed from bicistronic vectors.

Bicistronic vectors are useful tools for exogenous expression of two gene products from a single promoter element; however, reduced expression of protein from the second cistron compared with the first cistron is a common limitation to this approach. To overcome this limitation, we explored use of dihydrofolate reductase (DHFR) complementary DNA encoded in bicistronic vectors to induce a second protein of interest by methotrexate (MTX) treatment. Previous studies have demonstrated that levels of DHFR protein and DHFR fusion protein can be induced translationally following MTX treatment of cells. We demonstrated that in response to MTX treatment, DHFR partner protein in a bicistronic construct is induced for longer periods of time when compared with endogenous DHFR and DHFR fusion protein, in vitro and in vivo. Using rapamycin pretreatment followed by MTX treatment, we also devised a strategy to modulate levels of two proteins expressed from a bicistronic construct in a cap-independent manner. To our knowledge, this is the first report demonstrating that levels of proteins in DHFR-based bicistronic constructs can be induced and modulated using MTX and rapamycin treatment.

MicroRNA reexpression as differentiation therapy in cancer.

Since their discovery in the early 2000s, microRNAs (miRNAs) and their penchant for RNA interference have taken the scientific community by storm, working their way into virtually every corner of biological inquiry. The very nature of their action, the ability to simultaneously extinguish the expression of a multitude of genes and negate their functions, immediately suggested therapeutic promise. In this issue of the JCI, a step toward the realization of this promise is described. Taulli et al. demonstrate that the miRNAs miR-1/miR-206, which are routinely lost in advanced, poorly differentiated rhabdomyosarcoma (RMS) but characteristically expressed in the mature skeletal muscle from which these tumors arise, restore the myogenic differentiation program and block the tumorigenic phenotype (see the related article beginning on page 2366). Their data support the notion that these small RNAs, effectively functioning as "micro-sheriffs" by restoring myogenic law and order, hold substantial clinical potential as differentiation therapy for RMS and perhaps other solid tumors. miRNA reexpression therapy constitutes a novel approach to handcuff oncogenes and arrest tumor development.

Epigenetic reversal of acquired resistance to 5-fluorouracil treatment.

Acquired and intrinsic resistance still remains a limitation to the clinical use of 5-fluorouracil (5-FU). The contribution of epigenetic changes to the development of drug resistance remains to be elucidated. Several genes that are hypermethylated and silenced have been identified in colorectal cancer. Based on the findings described in the accompanying article, we hypothesized that acquired resistance to "pulse" 5-FU has an epigenetic origin and might be reversed. Here, we present a novel therapeutic approach to circumvent clinical resistance to bolus 5-FU, that is, treatment of bolus 5-FU-resistant colorectal cancer cells with low-dose 5-azadeoxycytidine (DAC), an inhibitor of DNA hypermethylation, restored sensitivity to 5-FU as well as 5-fluorouridine. Moreover, treatment of nude mice bearing a 5-FU-resistant tumor, characterized by decreased levels of UMP kinase (UMPK), with DAC overcame resistance to bolus 5-FU. DAC-mediated restoration of 5-FU sensitivity was associated with increases in UMPK levels. An increase in UMPK protein and mRNA levels following treatment with low-dose DAC was observed in cultured bolus 5-FU-resistant colorectal cancer cells (HCT-8) and in mice bearing these tumors. We conclude that DAC-mediated restoration of sensitivity to bolus 5-FU is mediated at least in part by increased UMPK levels and clinical resistance to 5-FU due to decreased UMPK in colorectal cancer may be overcome by including methylation inhibitors such as DAC.

Decreased levels of UMP kinase as a mechanism of fluoropyrimidine resistance.

5-Fluorouracil (5-FU) continues to be widely used for treatment of gastrointestinal cancers. Because many tumors show primary or acquired resistance, it is important to understand the molecular basis underlying the mechanism of resistance to 5-FU. In addition to its effect on thymidylate synthase inhibition and DNA synthesis, 5-FU may also influence RNA metabolism. Our previous studies revealed that colorectal cancer cells resistant to bolus 5-FU (HCT-8/4hFU) showed significantly decreased incorporation of the drug into RNA. Resistance to bolus 5-FU was associated with lower expression of UMP kinase (UMPK), an enzyme that plays an important role in the activation of 5-FU to 5-FUTP and its incorporation into RNA. Activities of other 5-FU-metabolizing enzymes (e.g., thymidine kinase, uridine phosphorylase, thymidine phosphorylase, and orotate phosphoribosyltransferase) remained unchanged between sensitive and resistant cell lines. Herein, we show that UMPK down-regulation in 5-FU-sensitive cells (HCT-8/P) induces resistance to bolus 5-FU treatment. Moreover, HCT-8/4hFU cells are even more cross-resistant to treatment with 5-fluorouridine, consistent with the current understanding of 5-fluorouridine as a RNA-directed drug. Importantly, colorectal cancer hepatic metastases isolated from patients clinically resistant to weekly bolus 5-FU/leucovorin treatment exhibited decreased mRNA expression of UMPK but not thymidylate synthase or dihydropyrimidine dehydrogenase compared with tumor samples of patients not previously exposed to 5-FU. Our findings provide new insights into the mechanisms of acquired resistance to 5-FU in colorectal cancer and implicate UMPK as an important mechanism of clinical resistance to pulse 5-FU treatment in some patients.

MicroRNA polymorphisms: the future of pharmacogenomics, molecular epidemiology and individualized medicine.

Referred to as the micromanagers of gene expression, microRNAs (miRNAs) are evolutionarily conserved small noncoding RNAs. Polymorphisms in the miRNA pathway (miR-polymorphisms) are emerging as powerful tools to study the biology of a disease and have the potential to be used in disease prognosis and diagnosis. Detection of miR-polymorphisms holds promise in the field of miRNA pharmacogenomics, molecular epidemiology and for individualized medicine. MiRNA pharmacogenomics can be defined as the study of miRNAs and polymorphisms affecting miRNA function in order to predict drug behavior and to improve drug efficacy. Advancements in the miRNA field indicate the clear involvement of miRNAs and genetic variations within the miRNA pathway in the progression and prognosis of diseases such as cancer, neurological disorders, muscular hypertrophy, gastric mucosal atrophy, cardiovascular disease and Type II diabetes. Various algorithms are available to predict miRNA-target mRNA sites; however, it is advisable to use multiple algorithms to confirm the predictions. Polymorphisms that may potentially affect miRNA-mediated regulation of the cell can be present not only in the 3 -UTR of a miRNA target gene, but also in the genes involved in miRNA biogenesis and in pri-, pre- and mature-miRNA sequences. A polymorphism in processed miRNAs may affect expression of several genes and have serious consequences, whereas a polymorphism in miRNA target site, in the 3 -UTR of the target mRNA, may be more target and/or pathway specific. In this review, we for the first time suggest a classification of miRNA polymorphisms/mutations. We also describe the importance and implications of miR-polymorphisms in gene regulation, disease progression, pharmacogenomics and molecular epidemiology.

Mesenchymal stem cells: flip side of the coin.

Tumor-associated fibroblasts or carcinoma-associated fibroblasts (CAF) play an important role in the growth of epithelial solid tumors. Although the cell type of origin of CAFs has not been conclusively established, it has been shown that they may be bone marrow derived. One side of the mesenchymal stem cell (MSC) coin is the well-accepted therapeutic potential of these cells for regenerative and immunomodulatory purposes. The ominous dark side is revealed by the recent work demonstrating that hMSCs may be a source of CAFs. In this review, we discuss the role of stromal cells in the tumor microenvironment and suggest that by exploring the in vitro/in vivo interplay between different cell types within the tumor milieu, strategies for improved tumor therapy can be developed.

Molecular targets for epigenetic therapy of cancer.

Recent advances in cancer research showed that changes of the cell "epigenome" contribute significantly to the development and progression of cancer. Similar to genetic alterations, epigenetic modifications can be transmitted to the next generation and used to turn off and/or on certain genes or pathways that may confer survival benefit to a malignant cell. However, epigenetic changes are readily reversible raising the possibility of "epigenetic therapy". A potential problem in this therapeutic approach is the lack of specificity, as epigenetic modifications are used by both normal and cancer cells to regulate expression of various genes. Ongoing studies to identify genes that are differentially expressed in cancer cells vs. normal cells are providing valuable information about molecular targets for epigenetic therapy. The present article will focus on summarizing some of these studies and will discuss the differences between conventional chemotherapy and epigenetic therapy utilizing epigenetic drugs like DNA methyltransferase inhibitors or histone deacetylase inhibitors. Current perspectives on the future of epigenetic therapy are also discussed.

MicroRNA polymorphisms: a giant leap towards personalized medicine.

"An individual's genetic inheritance of microRNA polymorphisms associated with disease progression, prognosis and treatment holds the key to create safer and more personalized drugs and can be a giant leap towards personalized medicine."