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Posts Tagged ‘microRNAs’


Regulatory MicroRNAs in Aberrant Cholesterol Transport and Metabolism

Curator: Marzan Khan, B.Sc

Aberrant levels of lipids and cholesterol accumulation in the body lead to cardiometabolic disorders such as atherosclerosis, one of the leading causes of death in the Western World(1). The physical manifestation of this condition is the build-up of plaque along the arterial endothelium causing the arteries to constrict and resist a smooth blood flow(2). This obstructive deposition of plaque is merely the initiation of atherosclerosis and is enriched in LDL cholesterol (LDL-C) as well foam cells which are macrophages carrying an overload of toxic, oxidized LDL(2). As the condition progresses, the plaque further obstructs blood flow and creates blood clots, ultimately leading to myocardial infarction, stroke and other cardiovascular diseases(2). Therefore, LDL is referred to as “the bad cholesterol”(2).

Until now, statins are most widely prescribed as lipid-lowering drugs that inhibit the enzyme 3-hydroxy-3methylgutaryl-CoA reductase (HMGCR), the rate-limiting step in de-novo cholesterol biogenesis (1). But some people cannot continue with the medication due to it’s harmful side-effects(1). With the need to develop newer therapeutics to combat cardiovascular diseases, Harvard University researchers at Massachusetts General Hospital discovered 4 microRNAs that control cholesterol, triglyceride, and glucose homeostasis(3)

MicroRNAs are non-coding, regulatory elements approximately 22 nucleotides long, with the ability to control post-transcriptional expression of genes(3). The liver is the center for carbohydrate and lipid metabolism. Stringent regulation of endogenous LDL-receptor (LDL-R) pathway in the liver is crucial to maintain a minimal concentration of LDL particles in blood(3). A mechanism whereby peripheral tissues and macrophages can get rid of their excess LDL is mediated by ATP-binding cassette, subfamily A, member 1 (ABCA1)(3). ABCA1 consumes nascent HDL particles- dubbed as the “good cholesterol” which travel back to the liver for its contents of triglycerides and cholesterol to be excreted(3).

Genome-wide association studies (GWASs) meta-analysis carried out by the researchers disclosed 4 microRNAs –(miR-128-1, miR-148a, miR-130b, and miR-301b) to lie close to single-nucleotide polymorphisms (SNPs) associated with abnormal metabolism and transport of lipids and cholesterol(3) Experimental analyses carried out on relevant cell types such as the liver and macrophages have proven that these microRNAs bind to the 3’ UTRs of both LDL-R and ABCA1 transporters, and silence their activity. Overexpression of miR-128-1 and miR148a in mice models caused circulating HDL-C to drop. Corroborating the theory under investigation further, their inhibition led to an increased clearance of LDL from the blood and a greater accumulation in the liver(3).

That the antisense inhibition of miRNA-128-1 increased insulin signaling in mice, propels us to hypothesize that abnormal expression of miR-128-1 might cause insulin resistance in metabolic syndrome, and defective insulin signaling in hepatic steatosis and dyslipidemia(3)

Further examination of miR-148 established that Liver-X-Receptor (LXR) activation of the Sterol regulatory element-binding protein 1c (SREBP1c), the transcription factor responsible for controlling  fatty acid production and glucose metabolism, also mediates the expression of miR-148a(4,5) That the promoter region of miR-148 contained binding sites for SREBP1c was shown by chromatin immunoprecipitation combined with massively parallel sequencing (ChIP-seq)(4). More specifically, SREBP1c attaches to the E-box2, E-box3 and E-box4 elements on miR-148-1a promoter sites to control its expression(4).

Earlier, the same researchers- Andres Naars and his team had found another microRNA called miR-33 to block HDL generation, and this blockage to reverse upon antisense targeting of miR-33(6).

These experimental data substantiate the theory of miRNAs being important regulators of lipoprotein receptors and transporter proteins as well as underscore the importance of employing antisense technologies to reverse their gene-silencing effects on LDL-R and ABCA1(4). Such a therapeutic approach, that will consequently lower LDL-C and promote HDL-C seems to be a promising strategy to treat atherosclerosis and other cardiovascular diseases(4).

References:

1.Goedeke L1,Wagschal A2,Fernández-Hernando C3, Näär AM4. miRNA regulation of LDL-cholesterol metabolism. Biochim Biophys Acta. 2016 Dec;1861(12 Pt B):. Biochim Biophys Acta. 2016 Dec;1861(12 Pt B):2047-2052

https://www.ncbi.nlm.nih.gov/pubmed/26968099

2.MedicalNewsToday. Joseph Nordgvist. Atherosclerosis:Causes, Symptoms and Treatments. 13.08.2015

http://www.medicalnewstoday.com/articles/247837.php

3.Wagschal A1,2, Najafi-Shoushtari SH1,2, Wang L1,2, Goedeke L3, Sinha S4, deLemos AS5, Black JC1,6, Ramírez CM3, Li Y7, Tewhey R8,9, Hatoum I10, Shah N11, Lu Y11, Kristo F1, Psychogios N4, Vrbanac V12, Lu YC13, Hla T13, de Cabo R14, Tsang JS11, Schadt E15, Sabeti PC8,9, Kathiresan S4,6,8,16, Cohen DE7, Whetstine J1,6, Chung RT5,6, Fernández-Hernando C3, Kaplan LM6,10, Bernards A1,6,16, Gerszten RE4,6, Näär AM1,2. Genome-wide identification of microRNAs regulating cholesterol and triglyceride homeostasis. . Nat Med.2015 Nov;21(11):1290

https://www.ncbi.nlm.nih.gov/pubmed/26501192

4.Goedeke L1,2,3,4, Rotllan N1,2, Canfrán-Duque A1,2, Aranda JF1,2,3, Ramírez CM1,2, Araldi E1,2,3,4, Lin CS3,4, Anderson NN5,6, Wagschal A7,8, de Cabo R9, Horton JD5,6, Lasunción MA10,11, Näär AM7,8, Suárez Y1,2,3,4, Fernández-Hernando C1,2,3,4. MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels. Nat Med. 2015 Nov;21(11):1280-9.

https://www.ncbi.nlm.nih.gov/pubmed/26437365

5.Eberlé D1, Hegarty B, Bossard P, Ferré P, Foufelle F. SREBP transcription factors: master regulators of lipid homeostasis. Biochimie. 2004 Nov;86(11):839-48.

https://www.ncbi.nlm.nih.gov/pubmed/15589694

6.Harvard Medical School. News. MicoRNAs and Metabolism.

https://hms.harvard.edu/news/micrornas-and-metabolism

7. MGH – Four microRNAs identified as playing key roles in cholesterol, lipid metabolism

http://www.massgeneral.org/about/pressrelease.aspx?id=1862

 

Other related articles published in this Open Access Online Scientific Journal include the following:

 

  • Cardiovascular Diseases, Volume Three: Etiologies of Cardiovascular Diseases: Epigenetics, Genetics and Genomics,

on Amazon since 11/29/2015

http://www.amazon.com/dp/B018PNHJ84

 

HDL oxidation in type 2 diabetic patients

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2015/11/27/hdl-oxidation-in-type-2-diabetic-patients/

 

HDL-C: Target of Therapy – Steven E. Nissen, MD, MACC, Cleveland Clinic vs Peter Libby, MD, BWH

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2014/11/07/hdl-c-target-of-therapy-steven-e-nissen-md-macc-cleveland-clinic-vs-peter-libby-md-bwh/

 

High-Density Lipoprotein (HDL): An Independent Predictor of Endothelial Function & Atherosclerosis, A Modulator, An Agonist, A Biomarker for Cardiovascular Risk

Curator: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/03/31/high-density-lipoprotein-hdl-an-independent-predictor-of-endothelial-function-artherosclerosis-a-modulator-an-agonist-a-biomarker-for-cardiovascular-risk/

 

Risk of Major Cardiovascular Events by LDL-Cholesterol Level (mg/dL): Among those treated with high-dose statin therapy, more than 40% of patients failed to achieve an LDL-cholesterol target of less than 70 mg/dL.

Reporter: Aviva Lev-Ari, PhD., RN

https://pharmaceuticalintelligence.com/2014/07/29/risk-of-major-cardiovascular-events-by-ldl-cholesterol-level-mgdl-among-those-treated-with-high-dose-statin-therapy-more-than-40-of-patients-failed-to-achieve-an-ldl-cholesterol-target-of-less-th/

 

LDL, HDL, TG, ApoA1 and ApoB: Genetic Loci Associated With Plasma Concentration of these Biomarkers – A Genome-Wide Analysis With Replication

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/12/18/ldl-hdl-tg-apoa1-and-apob-genetic-loci-associated-with-plasma-concentration-of-these-biomarkers-a-genome-wide-analysis-with-replication/

 

Two Mutations, in the PCSK9 Gene: Eliminates a Protein involved in Controlling LDL Cholesterol

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2013/04/15/two-mutations-in-a-pcsk9-gene-eliminates-a-protein-involve-in-controlling-ldl-cholesterol/

Artherogenesis: Predictor of CVD – the Smaller and Denser LDL Particles

Reporter: Aviva Lev-Ari, PhD, RN

https://pharmaceuticalintelligence.com/2012/11/15/artherogenesis-predictor-of-cvd-the-smaller-and-denser-ldl-particles/

 

A Concise Review of Cardiovascular Biomarkers of Hypertension

Curator: Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2016/04/25/a-concise-review-of-cardiovascular-biomarkers-of-hypertension/

 

Triglycerides: Is it a Risk Factor or a Risk Marker for Atherosclerosis and Cardiovascular Disease ? The Impact of Genetic Mutations on (ANGPTL4) Gene, encoder of (angiopoietin-like 4) Protein, inhibitor of Lipoprotein Lipase

Reporters, Curators and Authors: Aviva Lev-Ari, PhD, RN and Larry H. Bernstein, MD, FCAP

https://pharmaceuticalintelligence.com/2016/03/13/triglycerides-is-it-a-risk-factor-or-a-risk-marker-for-atherosclerosis-and-cardiovascular-disease-the-impact-of-genetic-mutations-on-angptl4-gene-encoder-of-angiopoietin-like-4-protein-that-in/

 

Excess Eating, Overweight, and Diabetic

Larry H Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2015/11/15/excess-eating-overweight-and-diabetic/

 

Obesity Issues

Larry H. Bernstein, MD, FCAP, Curator

https://pharmaceuticalintelligence.com/2015/11/12/obesity-issues/

 

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miRNA Therapeutic Promise

Curator: Larry H. Bernstein, MD, FCAP

 

MicroRNA Expression Could Be Key to Leukemia Treatment

http://www.genengnews.com/gen-news-highlights/microrna-expression-could-be-key-to-leukemia-treatment/81252662/

MicroRNA Expression Could Be Key to Leukemia Treatment

Generalized gene regulation mechanisms of miRNAs. [NIH]

 

Increasingly, cancer researchers are discovering novel biological pathways that regulate the expression of various genes that are often strongly associated with tumorigenesis. These new molecular mechanisms represent important potential therapeutic targets for aggressive and difficult-to-treat cancers. In particular, microRNAs (miRNAs)—small, noncoding genetic material that regulates gene expression—have steadily become implicated in the progression of some cancers.

Now, researchers at the University of Cincinnati (UC) have found a particular signaling route for a microRNA, miR-22, that they believe leads to targets for acute myeloid leukemia (AML), the most common type of fast-growing cancer of the blood and bone marrow.

The findings from this study were published recently in Nature Communications in an article entitled “miR-22 Has a Potent Anti-Tumour Role with Therapeutic Potential in Acute Myeloid Leukaemia.”

Structure of mi-22 miccroRNA. [Ppgardne at el., via Wikimedia Commons]

Increasingly, cancer researchers are discovering novel biological pathways that regulate the expression of various genes that are often strongly associated with tumorigenesis. These new molecular mechanisms represent important potential therapeutic targets for aggressive and difficult-to-treat cancers. In particular, microRNAs (miRNAs)—small, noncoding genetic material that regulates gene expression—have steadily become implicated in the progression of some cancers.

Now, researchers at the University of Cincinnati (UC) have found a particular signaling route for a microRNA, miR-22, that they believe leads to targets for acute myeloid leukemia (AML), the most common type of fast-growing cancer of the blood and bone marrow.

The findings from this study were published recently in Nature Communications in an article entitled “miR-22 Has a Potent Anti-Tumour Role with Therapeutic Potential in Acute Myeloid Leukaemia.”

“MicroRNAs make up a class of small, noncoding internal RNAs that control a gene’s job, or expression, by directing their target messaging RNAs, or mRNAs, to inhibit or stop. Cellular organisms use mRNA to convey genetic information,” explained senior study author Jianjun Chen, Ph.D., associate professor in the department of cancer biology at the UC College of Medicine. “Previous research has shown that microRNA miR-22 is linked to breast cancer and other blood disorders which sometimes turn into AML, but we found in this study that it could be an essential anti-tumor gatekeeper in AML when it is down-regulated, meaning its function is minimized.”

AML—most common type of acute leukemia—arises when the bone marrow begins to make blasts, cells that have not yet completely matured. These blast cells typically develop into white blood cells; however, in AML the cells do not develop and are unable to aid in warding off infections. In the current study, the UC team describes how altering the expression of miR-22 affected AML pathogenesis.

“When we forced miR-22 expression, we saw difficulty in leukemia cells developing, growing, and thriving. miR-22 targets multiple cancer-causing genes (CRTC1, FLT3, and MYCBP) and blocks certain pathways (CREB and MYC),” Dr. Chen noted. “The downregulation, or decreased output, of miR-22 in AML, is caused by the loss of the number of DNA being copied and/or stopping their expression through a pathway called TET1/GFI1/EZH2/SIN3A. Also, nanoparticles carrying miR-22 DNA oligonucleotides (short nucleic acid molecules) prevented leukemia advancement.”

The investigators conducted the study using bone marrow transplant samples and animal models. The researchers showed that the ten-eleven translocation proteins (TET1/2/3) in mammals helped to control genetic expression in normal developmental processes. This was in sharp contrast to mutations that cause function loss and tumor-slowing with TET2, which has been observed previously in blood and stem cell cancers.

“We recently reported that TET1 plays an essential cancer generating role in certain AML where it activates expression of homeobox genes, which are a large family of similar genes that direct the formation of many body structures during early embryonic development,” remarked Dr. Chen. “However, it is unknown whether TET1 can also function as a repressor for cellular function in cancer, and its role in microRNA expression has rarely been studied.”

Dr. Chen added that these findings are important in targeting a cancer that is both common and fatal, stating that “the majority of patients with ALM usually don’t survive longer than 5 years, even with chemotherapy, which is why the development of new effective therapies based on the underlying mechanisms of the disease is so important.”

“Our study uncovers a previously unappreciated signaling pathway (TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC) and provides new insights into genetic mechanisms causing and progressing AML and also highlights the clinical potential of miR-22-based AML therapy. More research on this pathway and ways to target it are necessary,” Dr. Chen concluded.

 

miR-22 has a potent anti-tumour role with therapeutic potential in acute myeloid leukaemia

Xi JiangChao HuStephen ArnovitzJason BugnoMiao YuZhixiang ZuoPing Chen, et al.
Nature Communications 26 Apr 2016; 7(11452).    http://dx.doi.org:/doi:10.1038/ncomms11452

MicroRNAs are subject to precise regulation and have key roles in tumorigenesis. In contrast to the oncogenic role of miR-22 reported in myelodysplastic syndrome (MDS) and breast cancer, here we show that miR-22 is an essential anti-tumour gatekeeper in de novo acute myeloid leukaemia (AML) where it is significantly downregulated. Forced expression of miR-22 significantly suppresses leukaemic cell viability and growth in vitro, and substantially inhibits leukaemia development and maintenance in vivo. Mechanistically, miR-22 targets multiple oncogenes, including CRTC1, FLT3 and MYCBP, and thus represses the CREB and MYC pathways. The downregulation of miR-22 in AML is caused by TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. Furthermore, nanoparticles carrying miR-22 oligos significantly inhibit leukaemia progression in vivo. Together, our study uncovers a TET1/GFI1/EZH2/SIN3A/miR-22/CREB-MYC signalling circuit and thereby provides insights into epigenetic/genetic mechanisms underlying the pathogenesis of AML, and also highlights the clinical potential of miR-22-based AML therapy.

 

As one of the most common and fatal forms of hematopoietic malignancies, acute myeloid leukaemia (AML) is frequently associated with diverse chromosome translocations (for example t(11q23)/MLL-rearrangements, t(15;17)/PML-RARA and t(8;21)/AML1-ETO) and molecular abnormalities (for example, internal tandem duplications of FLT3 (FLT3-ITD) and mutations in nucleophosmin (NPM1c+))1. Despite intensive chemotherapies, the majority of patients with AML fail to survive longer than 5 years2, 3. Thus, development of effective therapeutic strategies based on a better understanding of the molecular mechanisms underlying the pathogenesis of AML is urgently needed.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that post-transcriptionally regulate gene expression4. Individual miRNAs may play distinct roles in cancers originating from different tissues or even from different lineages of hematopoietic cells4. It is unclear whether a single miRNA can play distinct roles between malignancies originating from the same hematopoietic lineage, such as de novo AML and myelodysplastic syndrome (MDS). Although around 30% of MDS cases transform to AML, the genetic and epigenetic landscapes of MDS or MDS-derived AML are largely different from those of de novo AML5, 6. MDS and MDS-derived AML are more responsive to hypomethylating agents than de novo AML7. The molecular mechanisms underlying the distinct pathogenesis and drug response between MDS (or MDS-derived AML) and de novo AML remain unclear.

The ten-eleven translocation (Tet1/2/3) proteins play critical transcriptional regulatory roles in normal developmental processes as activators or repressors8, 9, 10. In contrast to the frequent loss-of-function mutations and tumour-suppressor role of TET2 observed in hematopoietic malignancies11, 12, 13, we recently reported that TET1 plays an essential oncogenic role in MLL-rearranged AML where it activates expression of homeobox genes14. However, it is unknown whether TET1 can also function as a transcriptional repressor in cancer. Moreover, Tet1-mediated regulation of miRNA expression has rarely been studied10.

In the present study, we demonstrate that miR-22, an oncogenic miRNA reported in breast cancer and MDS15, 16, is significantly downregulated in most cases of de novo AML due to TET1/GFI1/EZH2/SIN3A-mediated epigenetic repression and/or DNA copy-number loss. miR-22 functions as an essential anti-tumour gatekeeper in various AML and holds great therapeutic potential to treat AML.

 

The downregulation of miR-22 in de novo AML

Through Exiqon miRNA array profiling, we previously identified a set of miRNAs, such as miR-150, miR-148a, miR-29a, miR-29b, miR-184, miR-342, miR-423 and miR-22, which are significantly downregulated in AML compared with normal controls17. Here we showed that among all the above miRNAs, miR-150 and especially miR-22 exhibited the most significant and consistent inhibitory effect on MLL-AF9-induced cell immortalization in colony-forming/replating assays (CFA) (Supplementary Fig. 1a). In contrast to the reported upregulation of miR-22 in MDS16, our original microarray data17 (Fig. 1a,b) and new quantitative PCR-independent validation data (Supplementary Fig. 1b) demonstrated a significant and global downregulation of miR-22 in de novo AML relative to normal controls. Notably, miR-22 is significantly downregulated in AML samples (P<0.05) compared with all three sub-populations of normal control cells, that is, normal CD34+ hematopoietic stem/progenitor cells (HSPCs), CD33+ myeloid progenitor cells, or mononuclear cells (MNCs) (Fig. 1a). Expression of miR-22 is significantly downregulated in all or the majority of individual subsets of AML samples than in the normal CD33+ or CD34+ cell samples (Fig. 1b).

Figure 1: miR-22 inhibits AML cell transformation and leukemogenesis.

miR-22 inhibits AML cell transformation and leukemogenesis.

(a,b) Exiqon microRNA profiling assay showed that miR-22 is significantly (P<0.05) downregulated in the entire set of AML set (n=85) (a) or each individual subset (b), relative to normal controls. The expression data were log(2) transformed and mean-centred. Mean±s.e.m. values were shown. (c) Comparison of effects of in-house miR-22, miR-22_Song16 and miR-22 mutant (miR-22mut; see the mutation sequence at the top) on MLL-AF9-induced colony forming. CFAs were performed using mouse BM progenitor (Lin) cells transduced with MSCV-neo+MSCV-PIG (Ctrl), MSCV-neo-MLL-AF9+MSCV-PIG (MLL-AF9), or MSCV-neo-MLL-AF9+MSCV-PIG-miR-22/miR-22_Song/miR-22mut. (d) Effects of miR-22 on the colony forming induced by multiple fusion genes. CFA was performed using wild-type BM progenitor cells co-transduced with MSCV-neo-MLL-AF9 (MA9), -MLL-AF10 (MA10), -PML-RARA (PR) or –AML1-ETO9a(AE9a)19, together with MSCV-PIG (Ctrl) or MSCV-PIG-miR-22 (+miR-22), as well as miR-22−/− BM progenitors co-transduced with individual fusion genes and MSCV-PIG. Colony counts (mean±s.d.) of the second round of plating are shown. *P<0.05; **P<0.01. (e,f) Effect of miR-22 on MLL-AF9-induced primary leukemogenesis. Kaplan–Meier curves are shown for six cohorts of transplanted mice including MSCVneo+MSCV-PIG (Ctrl; n=5), MSCVneo+MSCV-PIG-miR-22 (miR-22; n=5), MSCVneo-MLL-AF9+MSCV-PIG (MA9; n=8), MSCVneo-MLL-AF9+MSCV-PIG-miR-150 (MA9+miR-150, n=6), MSCVneo-MLL-AF9+MSCV-PIG-miR-22 (MA9+miR-22; n=10) and MSCVneo-MLL-AF9+MSCV-PIG-miR-22mutant (MA9+miR-22mut; n=5) (e); Wright–Giemsa stained PB and bone marrow (BM), and hematoxylin and eosin (H&E) stained spleen and liver of the primary BMT recipient mice at the end point are shown (f). (g) Effect of miR-22 on MLL-AF10-induced primary leukemogenesis. Kaplan–Meier curves are shown for two cohorts of transplanted mice including MSCVneo-MLL-AF10+MSCV-PIG (MA10; n=5) and MSCVneo-MLL-AF10+MSCV-PIG-miR-22 (MA10+miR-22; n=5). (h) miR-22 knockout promotes AE9a-induced leukemogenesis. Kaplan–Meier curves are shown for mice transplanted with wild-type or miR-22−/− BM progenitor cells transduced MSCV-PIG-AE9a (n=5 for each group). The P values were generated by t-test (ad) or log-rank test (e,g,h).

To rule out the possibility that the inhibitory effect of miR-22 shown in Supplementary Fig. 1a was due to a non-specific effect of our miR-22 construct, we included the MSCV-PIG-miR-22 construct from Song et al.16 in a repeated CFA. Both miR-22 constructs dramatically inhibited MLL-AF9-induced colony formation (Fig. 1c). As the ‘seed’ sequences at the 5′ end of individual miRNAs are essential for the miRNA-target binding18, we also mutated the 6-bases ‘seed’ sequence of miR-22 and found that the miR-22 mutant did not inhibit colony formation anymore (Fig. 1c). In human AML cells, forced expression of miR-22, but not miR-22 mutant, significantly inhibited cell viability and growth/proliferation, while promoting apoptosis (Supplementary Fig. 1c,d).

Furthermore, as miR-22 is globally downregulated in all major types of AML (Fig. 1b), we also investigated the role of miR-22 in colony formation induced by other oncogenic fusion genes, including MLL-AF10/t(10;11), PML-RARA/t(15;17) and AML1-ETO9a/t(8;21) (ref. 19). As expected, forced expression of miR-22 significantly inhibited colony formation induced by all individual oncogenic fusions; conversely, miR-22 knockout20 significantly enhanced colony forming (Fig. 1d). These results suggest that miR-22 likely plays a broad anti-tumour role in AML.

In accordance with the potential anti-tumour function of miR-22 in AML, miR-22 was expressed at a significantly higher level (P<0.05) in human normal CD33+ myeloid progenitor cells than in more immature CD34+ HSPCs or MNC cells (a mixed population containing both primitive progenitors and committed cells) (Fig. 1a,b), implying that miR-22 is upregulated during normal myelopoiesis. Similarly, we showed that miR-22 was also expressed at a significantly higher level in mouse normal bone marrow (BM) myeloid (Gr-1+/Mac-1+) cells, relative to lineage negative (Lin) progenitor cells, long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), and committed progenitors (CPs) (Supplementary Fig. 1e), further suggesting that miR-22 is upregulated in normal myelopoiesis.

The anti-tumour effect of miR-22 in the pathogenesis of AML

Through bone marrow transplantation (BMT) assays, we showed that forced expression of miR-22 (but not miR-22 mutant) dramatically blocked MLL-AF9 (MA9)-mediated leukemogenesis in primary BMT recipient mice, with a more potent inhibitory effect than miR-150 (Fig. 1e;Supplementary Fig. 2a). All MA9+miR-22 mice exhibited normal morphologies in peripheral blood (PB), BM, spleen and liver tissues (Fig. 1f), with a substantially reduced c-Kit+ blast cell population in BM (Supplementary Fig. 2b). Forced expression of miR-22 also almost completely inhibited leukemogenesis induced by MLL-AF10 (Fig. 1g; Supplementary Fig. 2a). Conversely, miR-22 knockout significantly promoted AML1-ETO9a (AE9a)-induced AML (Fig. 1h). Thus, the repression of miR-22 is critical for the development of primary AML. Notably, forced expression of miR-22 inMLL-AF9 and MLL-AF10 leukaemia mouse models caused only a 2–3-fold increase in miR-22 expression level (Supplementary Fig. 2a), in a degree comparable to the difference in miR-22 expression levels between human AML samples and normal controls (Fig. 1a), suggesting that a 2–3-fold change in miR-22 expression level appears to be able to exert significant physiological or pathological effects.

To examine whether the maintenance of AML is also dependent on the repression of miR-22, we performed secondary BMT assays. Forced expression of miR-22 remarkably inhibited progression of MLL-AF9-, AE9a– or FLT3-ITD/NPM1c+-induced AML in secondary recipient mice (Fig. 2a–d), resulting in largely normal morphologies in PB, BM, spleen and liver tissues (Fig. 2b;Supplementary Fig. 2c). Collectively, our findings demonstrate that miR-22 is a pivotal anti-tumour gatekeeper in both development and maintenance of various AML.

Figure 2: Effect of miR-22 on the maintenance of AML in vivo.

Effect of miR-22 on the maintenance of AML in vivo.

(a,b) Effect of miR-22 on the maintenance of MLL-AF9-induced AML in secondary BMT recipient mice. The secondary BMT recipients were transplanted with BM blast cells from the primary MLL-AF9 AML mice retrovirally transduced with MSCV-PIG+MSCVneo (MA9-AML+Ctrl; n=7) or MSCV-PIG+MSCVneo-miR-22 (MA9-AML+miR-22; n=10). Kaplan–Meier curves (a) and Wright–Giemsa or H&E-stained PB, BM, spleen and liver (b) of the secondary leukaemic mice are shown. (c,d) Effect of miR-22 on the maintenance/progression of AML1-ETO9a (AE9a)-induced AML (c) or FLT3-ITD/NPM1c+-induced AML (d) in secondary BMT recipient mice (n=5 for each group). Kaplan–Meier curves and P values (log-rank test) are shown.

 

Identification of critical target genes of miR-22 in AML

To identify potential targets of miR-22 in AML, we performed a series of data analysis. Analysis of In-house_81S (ref. 21) and TCGA_177S (ref. 22) data sets revealed a total of 999 genes exhibiting significant inverse correlations with miR-22 in expression. Of them, 137 genes, including 21 potential targets of miR-22 as predicted by TargetScan18 (Supplementary Table 1), were significantly upregulated in both human and mouse AML compared with normal controls as detected in two additional in-house data sets14, 23. Among the 21 potential targets, CRTC1, ETV6and FLT3 are known oncogenes24, 25, 26, 27, 28, 29. We then focused on these three genes, along with MYCBP that encodes the MYC-binding protein and is an experimentally validated target of miR-22 (ref. 30) although due to a technical issue it was not shown in the 21-gene list (Supplementary Table 1), for further studies.

As expected, all four genes were significantly downregulated in expression by ectopic expression of miR-22 in human MONOMAC-6/t(9;11) cells (Fig. 3a). The coincidence of downregulation of those genes and upregulation of miR-22 was also observed in mouse MLL-ENL-ERtm cells, a leukaemic cell line with an inducible MLL-ENL derivative31, when MLL-ENL was depleted by 4-hydroxy-tamoxifen (4-OHT) withdrawal (Fig. 3b; Supplementary Fig. 3a). While MLL-AF9 remarkably promoted expression of those four genes in mouse BM progenitor cells, co-expressed miR-22 reversed the upregulation (Fig. 3c). In leukaemia BM blast cells of mice with MLL-AF9-induced AML, the expression of Crtc1, Flt3 and Mycbp, but not Etv6, was significantly downregulated by co-expressed miR-22 (but not by miR-22 mutant) (Fig. 3d). Because miR-22-mediated downregulation of Etv6 could be observed only in the in vitro models (Fig. 3a–c), but not in the in vivo model (Fig. 3d), which was probably due to the difference between in vitro and in vivo microenvironments, we decided to focus on the three target genes (that is, Crtc1, Flt3 and Mycbp) that showed consistent patterns between in vitro and in vivo for further studies. The repression of Crtc1, Flt3 and Mycbpwas also found in leukaemia BM cells of mice with AE9a or FLT3-ITD/NPM1c+-induced AML (Fig. 3e,f). As Mycbp is already a known target of miR-22 (ref. 30), here we further confirmed that FLT3and CRTC1 are also direct targets of miR-22 (Fig. 3g,h). The downregulation of CRTC1, FLT3 and MYCBP by miR-22 at the protein level was confirmed in both human and mouse leukaemic cells (Supplementary Fig. 3b,c). Overexpression of miR-22 had no significant influence on the level of leukaemia fusion genes (Supplementary Fig. 3d).

Figure 3: miR-22 targets multiple oncogenes.

miR-22 targets multiple oncogenes.

(a) Downregulation of CRTC1, FLT3, MYCBP and ETV6 by forced expression of miR-22 in MONOMAC-6 cells. Expression of these genes was detected 48h post transfection of MSCV-PIG (Ctrl) or MSCV-PIG-miR-22 (miR-22). (b) Crtc1, Flt3, Mycbp and Etv6 levels in MLL-ENL-ERtm cells after withdrawal of 4-OHT for 0, 7 or 10 days. (c) Expression levels of Crtc1, Flt3, Mycbp and Etv6 in mouse BM progenitor cells retrovirally transduced with MSCV-PIG+MSCV-neo (Ctrl), MSCV-PIG-miR-22+MSCV-neo (miR-22), MSCV-PIG+MSCV-neo-MLL-AF9 (MLL-AF9) or MSCV-PIG-miR-22+MSCV-neo-MLL-AF9 (MLL-AF9+miR-22). (d) Expression levels of Crtc1, Flt3, Mycbp and Etv6 in BM blast cells of leukaemic mice transplanted with MLL-AF9, MLL-AF9+miR-22 or MLL-AF9+miR-22mut primary leukaemic cells. (e,f) Expression levels of Crtc1, Flt3 and Mycbp in BM blast cells of leukaemic mice transplanted with MSCV-PIG or MSCV-PIG-miR-22-retrovirally transduced AE9a (e) or FLT3-ITD/NPM1c+ (f) primary leukaemic cells. (g) Putative miR-22 target sites and mutants in the 3′UTRs of CRTC1 (upper panel) and FLT3(lower panel). (h) Effects of miR-22 on luciferase activity of the reporter gene bearing wild type or mutant 3′UTRs of CRTC1 or FLT3 in HEK293T cells. The mean±s.d. values from three replicates are shown.*P<0.05, t-test.

Co-expression of the coding region (CDS) of each of the three target genes (that is, CRTC1, FLT3and MYCBP) largely reversed the effects of miR-22 on cell viability, apoptosis and proliferation (Fig. 4a–e). More importantly, in vivo BMT assays showed that co-expressing CRTC1, FLT3 orMYCBP largely rescued the inhibitory effect of miR-22 on leukemogenesis (Fig. 4f,g;Supplementary Fig. 3e). Our data thus suggest that CRTC1, FLT3 and MYCBP are functionally important targets of miR-22 in AML.

Figure 4: Multiple onocgenes are functionally important targets of miR-22 in AML.

Multiple onocgenes are functionally important targets of miR-22 in AML.

(a,b) Relative viability (a) and apoptosis (b) levels of MONOMAC-6 cells transfected with MSCV-PIG-CRTC1, -FLT3 or –MYCBP alone, or together with MSCVneo-miR-22. Values were detected 48h post transfection. (c–e) Rescue effects of CRTC1 (c), FLT3 (d) and MYCBP (e) on the inhibition of MONOMAC-6 growth mediated by miR-22. Cell counts at the indicated time points are shown. Mean±s.d. values are shown. *P<0.05, t-test. (f) In vivo rescue effects of CRTC1, FLT3 and MYCBP on the inhibition of MLL-AF9-induced leukemogenesis mediated by miR-22. The secondary recipients were transplanted with BM blast cells of the primary MLL-AF9 leukaemic mice retrovirally transduced with MSCVneo+MSCV-PIG (MA9-AML+Ctrl; n=7), MSCVneo-miR-22+MSCV-PIG (MA9-AML+miR-22; n=10), MSCVneo-miR-22+MSCV-PIG-CRTC1 (MA9-AML+miR-22+CRTC1; n=5), MSCVneo-miR-22+MSCV-PIG-FLT3 (MA9-AML+miR-22+FLT3; n=6) or MSCVneo-miR-22+MSCV-PIG-MYCBP (MA9-AML+miR-22+MYCBP; n=6). Kaplan–Meier curves for all the five groups of transplanted mice are shown. MA9-AML+Ctrl versus MA9-AML+miR-22, P<0.001 (log-rank test); MA9-AML+Ctrl versus any other groups,P>0.05 (log-rank test). (g) Wright–Giemsa stained PB and BM, and H&E stained spleen and liver of the secondary leukaemic mice.

miR-22 represses both CREB and MYC signalling pathways

DNA copy-number loss of miR-22 gene locus in AML

Expression of miR-22 is epigenetically repressed in AML

 

Figure 5: Transcriptional correlation between miR-22 and TET1.

(a) Correlation between the expression levels of miR-22 and TET1 in three independent AML patient databases. All expression data were log(2) transformed; the data in In-house_81S were also mean-centred. The correlation coefficient (r) and P values were detected by ‘Pearson Correlation’, and the correlation regression lines were drawn with the ‘linear regression’ algorithm. (b) Expression of pri-, pre- and mature miR-22, and Tet1/2/3 in colony-forming cells of wild-type mouse BM progenitors retrovirally transduced with MSCVneo (Ctrl), MSCVneo-MLL-AF9 (MLL-AF9), MSCVneo-MLL-AF10 (MLL-AF10) or MSCVneo-AE9a (AE9a), or of FLT3-ITD/NPM1c+ mouse BM progenitors transduced with MSCVneo (FLT3-ITD+/NPM1c+). (c) Expression of miR-22 and Tet1/2/3 in MLL-ENL-ERtm cells. Expression levels were detected at the indicated time points post 4-OHT withdrawal. (d) Effect of miR-22 overexpression onTet1 expression in colony-forming cells with MLL-AF9, AE9a or FLT3-ITD/NPM1c+. (e) Expression ofTet1 in BM progenitor cells of 6-weeks old miR-22−/− or wild-type mice. (f) Effect of miR-22 overexpression on TET1 expression in THP-1 and KOCL-48 AML cells 48h post transfection. (g) Expression of pri-, pre- and mature miR-22 in BM progenitor cells of 6-weeks old Tet1−/− or wild-type mice. Mean±s.d. values are shown. *P<0.05, t-test.

(a) Tet1 targets miR-22 promoter region (−1,100/+55bp), as detected by luciferase reporter assay 48h post transfection in HEK293T cells. (b) Expression of TET1/2/3, EZH2, SIN3A, GFI1 and miR-22 in THP-1 cells 72h post treatment with 1μM ATRA or DMSO control. (c) Co-immunoprecipitation assay showing the binding of endogenous GFI1 and TET1 in THP1 cells. (d) ChIP-qPCR analyses of the promoter region of miR-22 in THP-1 cells 72h post treatment with 1μM ATRA or DMSO. Upper panel: PCR site on the CpG-enriched region of miR-22 gene locus. Note: miR-22 is coded within the second exon of a long non-coding RNA (MIR22HG), which represents the primary transcript of miR-22. Lower panels: enrichment of MLL-N terminal (for both wild-type MLL and MLL-fusion proteins), MLL-C terminal (for wild-type MLL), TET1, EZH2, SIN3A, GFI1, H3K27me3, H3K4me3 or RNA pol II at miR-22 promoter region. (e) Expression levels of TET1, EZH2, SIN3A and miR-22 in GFI1 knockdown cells. (f) ChIP-qPCR analyses of the promoter region of miR-22 in THP-1 cells transduced with GFI1 shRNA or control shRNA. Enrichment of GFI1, TET1, EZH2 and SIN3A are shown. (g) Effects of knockdown of TET1, EZH2 and/orSIN3A on miR-22 expression. The expression level of miR-22 was detected in THP-1 cells 72h post transfection with siRNAs targeting TET1, EZH2 and/or SIN3A. Mean±s.d. values are shown. *P<0.05;**P<0.01 (t-test). (h) Schematic model of the regulatory pathway involving miR-22 in AML and ATRA treatment.

 

The miR-22-associated regulatory circuit in AML

         Restoration of miR-22 expression and function to treat AML

 

Figure 7: Therapeutic effect of miR-22-nanoparticles in treating AML.

(a,b) Primary leukaemia BM cells bearing MLL-AF9 (a) or AE9a (b) were transplanted into sublethally irradiated secondary recipient mice. After the onset of secondary AML (usually 10 days post transplantation), the recipient mice were treated with PBS control, or 0.5mgkg−1 miR-22 or miR-22 mutant RNA oligos formulated with G7 PAMAM dendrimer nanoparticles, i.v., every other day, until the PBS-treated control group all died of leukaemia. (c) NSGS mice49 were transplanted with MV4;11/t(4;11) AML cells. Five days post transplantation, these mice started to be treated with PBS control, miR-22 or miR-22 mutant nanoparticles at the same dose as described above. Kaplan–Meier curves are shown; the drug administration period and frequency were indicated with yellow arrows. The P values were detected by log-rank test. (d) Wright–Giemsa stained PB and BM, and H&E stained spleen and liver of the MLL-AF9-secondary leukaemic mice treated with PBS control, miR-22 or miR-22 mutant nanoparticles.

We then tested the miR-22 nanoparticles in a xeno-transplantation model49. Similarly, the nanoparticles carrying miR-22 oligos, but not miR-22 mutant, significantly delayed AML progression induced by human MV4;11/t(4;11) cells (Fig. 7c). The miR-22-nanoparticle administration also resulted in less aggressive leukaemic pathological phenotypes in the recipient mice (Supplementary Fig. 6e). Thus, our studies demonstrated the therapeutic potential of using miR-22-based nanoparticles to treat AML.

 

It remains poorly understood how TET proteins mediate gene regulation in cancer. Here we show that in de novo AML, it is TET1, but not TET2 (a reported direct target of miR-22 in MDS and breast cancer15, 16), that inversely correlates with miR-22 in expression and negatively regulates miR-22 at the transcriptional level. Likely together with GFI1, TET1 recruits polycomb cofactors (for example, EZH2/SIN3A) to the miR-22 promoter, leading to a significant increase in H3K27me3 occupancy and decrease in RNA pol II occupancy at that region, and thereby resulting in miR-22 repression in AML cells; such a repression can be abrogated by ATRA treatment. Thus, our study uncovers a novel epigenetic regulation mechanism in leukaemia involving the cooperation between TET1/GFI1 and polycomb factors.

Besides GFI1, it was reported that LSD1 is also a binding partner of TET1 (ref. 50). Interestingly, LSD1 is known as a common binding partner shared by TET1 and GFI1, and mediates the effect of GFI1 on hematopoietic differentiation51, 52. Thus, it is possible that LSD1 might also participate in the transcriptional repression of miR-22 as a component of the GFI1/TET1 repression complex.

We previously reported that TET1 cooperates with MLL fusions in positively regulating their oncogenic co-targets in MLL-rearranged AML14. Here we show that TET1 can also function as a transcriptional repressor (of a miRNA) in cancer. The requirement of TET1-mediated regulation on expression of its positive (for example, HOXA/MEIS1/PBX3)14 or negative (for example, miR-22) downstream effectors in leukemogenesis likely explains the rareness of TET1 mutations in AML53, and highlights its potent oncogenic role in leukaemia.

The aberrant activation of both CREB and MYC signalling pathways has been shown in AML24, 25,26, 54, 55, but the underlying molecular mechanisms remain elusive. Our data suggest that the activation of these two signalling pathways in AML can be attributed, at least in part, to the repression of miR-22, which in turn, results in the de-repression of CRTC1 (CREB pathway), FLT3and MYCBP (MYC pathway), and leads to the upregulation of oncogenic downstream targets (for example, CDK6, HOXA7, BMI1, FASN and HMGA1) and downregulation of tumour-suppressor downstream targets (for example, RGS2).

In summary, we uncover a TET1/GFI1/EZH2/SIN3A⊣miR-22⊣CREB-MYC signalling circuit in de novo AML, in which miR-22 functions as a pivotal anti-tumour gate-keeper, distinct from its oncogenic role reported in MDS or MDS-derived AML16. Thus, our study together with the study of Song et al.16 highlight the complexity and functional importance of miR-22-associated gene regulation and signalling pathways in hematopoietic malignancies, and may provide novel insights into the genetic/epigenetic differences between de novo AML and MDS.

Our findings also highlight the possibility of using miR-22-based therapy to treat AML patients. Our proof-of-concept studies demonstrate that the nanoparticles carrying miR-22 oligos significantly inhibit AML progression and prolong survival of leukaemic mice in both BMT and xeno-transplantation models. Notably, miRNA-based nanoparticles have already entered clinical trials56. It would be important, in the future, to further test the combination of miR-22-carrying nanoparticles (or small-molecule compounds that can induce endogenous expression of miR-22) with standard chemotherapy agents (cytosine arabinoside and anthracycline), or with the emerging small molecule inhibitors against MYC and/or CREB pathway effectors, to achieve optimal anti-leukaemia effect with minimal side effects. Overall, our results suggest that restoration of miR-22 expression/function (for example, using miR-22-carrying nanoparticles or small-molecule compounds) holds great therapeutic potential to treat AML, especially those resistant to current therapies.

 

MicroRNAs: A Gene Silencing Mechanism with Therapeutic Implications  

Wed, July 13, 2016   The New York Academy of Sciences    Presented by the Biochemical Pharmacology Discussion Group
http://www.nyas.org/Events/Detail.aspx?cid=787a5d77-8354-4df7-92d5-91db18b2ce49

MicroRNAs (miRNAs) are single-stranded RNAs about 22 nucleotides in length that repress the expression of specific proteins by annealing to complementary sequences in the 3′ untranslated regions (UTRs) of target mRNAs. Apart from their posttranscriptional expression, or silencing, miRNAs may also direct mRNA destabilization and cleavage. Moreover, rather than targeting a single disease-associated protein target as many small molecule drugs and antibodies do, each miRNA may serve to repress the expression of numerous proteins involved in the pathogenesis and progression of various diseases and could therefore potentially interfere with multiple disease-promoting signal transduction pathways. Because aberrant expression of miRNAs has been implicated in numerous disease states, miRNA-based therapies have sparked much interest for the treatment of a variety of diseases. The objective of this symposium is to bring together investigators who have led the field in describing what miRNAs do and their potential in treating diseases, as well as those who are translating these findings into promising drug candidates, some of which have already advanced into early stage clinical trials.

Call for Poster Abstracts

Abstract submissions are invited for a poster session. For complete submission instructions, please send an email to miRNA@nyas.org with the words “Abstract Information” in the subject line. The deadline for abstract submission is May 13, 2016.

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Prognostic biomarker for NSCLC and Cancer Metastasis

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Membranous CD24 expression as detected by the monoclonal antibody SWA11 is a prognostic marker in non-small cell lung cancer patients

Michael MajoresAnne SchindlerAngela FuchsJohannes SteinLukas HeukampPeter Altevogt and Glen Kristiansen

BMC Clinical Pathology201515:19   http://dx.doi.org:/10.1186/s12907-015-0019-z

Background    Lung cancer is one of the most common malignant neoplasms worldwide and has a high mortality rate. To enable individualized therapy regimens, a better understanding of the molecular tumor biology has still to be elucidated. The expression of the cell surface protein CD24 has already been claimed to be associated with shorter patient survival in non-small cell lung cancer (NSCLC), however, the prognostic value and applicability of CD24 immunostaining in paraffin embedded tissue specimens has been questioned due to the recent acknowledgement of restricted epitope specificity of the commonly used antibody SN3b.   Methods    A cohort of 137 primary NSCLC cases was immunostained with a novel CD24 antibody (clone SWA11), which specifically recognizes the CD24 protein core and the resulting expression data were compared with expression profiles based on the monoclonal antibody SN3b. Furthermore, expression data were correlated to clinico-pathological parameters. Univariate and multivariate survival analyses were conducted with Kaplan Meier estimates and Cox regression, respectively. Results    CD24 positivity was found in 34 % resp. 21 % (SN3b) of NSCLC with a membranous and/or cytoplasmic staining pattern. Kaplan-Meier analyses revealed that membranous, but not cytoplasmic CD24 expression (clone SWA11) was associated with lympho-nodular spread and shorter overall survival times (both p < 0.05). CD24 expression established by SN3b antibodies did not reveal significant clinicopathological correlations with overall survival, neither for cytoplasmic nor membranous CD24 staining.  Conclusions    Membranous CD24 immunoreactivity, as detected with antibody clone SWA11 may serve as a prognostic factor for lymphonodular spread and poorer overall survival. Furthermore, these results corroborate the importance of a careful distinction between membranous and cytoplasmic localisation, if CD24 is to be considered as a potential prognostic biomarker.

 

Lung cancer is a major cause of carcinoma related death, being responsible for 17.8 % of all cancer deaths and accounting for more than a million deaths worldwide per year [1]. Despite intense studies to improve therapy options, its prognosis has remained poor with a 5-year overall survival rate of less than 15 % [2].

In the past decade, the largest subgroup of lung cancer, i.e. non-small cell lung cancer (NSCLC), has been subjected to exerted research for a better understanding of the underlying molecular biology of lung cancer. More than ten years ago, CD24 has already been suggested as a novel and promising biomarker for carcinoma progression in NSCLC [3] and several groups have confirmed this finding on protein and transcript level [2, 4]. CD24 is a highly glycosylated protein, that binds to the cell surface through a GPI (glycosyl-phosphatidylinositol)-anchor and functions as a cell adhesion molecule and is involved in cell-cell-interaction via its P-selectin binding site [5]. CD24 has been found to be expressed by pre-B-lymphocytes [5]. It is assumed that CD24-positive cells can attach more easily to platelets and activated endothelial cells [6, 7]. Notably, CD24 has also been observed in many human carcinomas, such as ovarian cancer, renal cell cancer, breast cancer and NSCLC [3, 812]. In epithelial ovarian cancer high scores of cytoplasmic CD24 were highly predictive of shorter patient survival times (mean 97.8 vs. 36.5 months), whereas membranous CD24 expression seemed to have no influence on survival times. Interestingly, CD24 positivity (membranous or cytoplasmic) of prostate cancer samples was significantly associated to younger patient age and higher pT stages and a higher 3-year prostate-specific antigen (PSA) relapse rate compared with CD24-negative tumours.

In patients with gallbladder carcinoma, tumors with up-regulation of CD24 revealed lymph node metastasis and lymphovascular invasion more frequently. Moreover, up-regulation of CD24 tended to show deeper invasion depth and higher TNM stage [13]. Together, these findings support CD24 as a prognostic marker for carcinoma progression and poorer survival.

Despite these intriguing findings, major concerns regarding a lack of epitope specificity of the commonly used monoclonal antibody SN3b have been raised [14]. Recent findings indicate that the mAb (monoclonal antibody) SN3b does not bind to the protein core itself, but binds to a glycan structure that decorates the CD24 molecule. On the one hand, this motif is not present on all forms of CD24 and—on the other hand—it can be present in other epitopes irrespective of CD24 [14]. These limitations underline the need for more specific CD24 antibodies, such as the mAb SWA11 antibody that has been suggested to be more specific as it binds to the protein core [14].

As CD24 is a promising biomarker for the risk assessment of disease progression, the goal of the present study was to investigate CD24 expression in NSCLC using the novel, more specific monoclonal antibody (mAb) SWA11. Special emphasis was put on the comparison of SN3b- and SWA11-mediated CD24 detection regarding a) the subcellular distribution of CD24 expression (i.e. membranous versus cytoplasmic expression) and b) its correlation with various clinicopathological features including patient survival times.

Table 1

Clinicopathological characteristics of the NSCLC cohort

  AC SCC
N (%) N (%)
Tumour stage (pT)
1 29 (21.2 %) 5 (3.6)
2 51 (37.2 %) 23 (16.8 %)
3 6 (4.4 %) 6 (4.4 %)
4 1 (0.7 %) 0 (0 %)
Nodal Status (pN) 0 37 (27.0 %) 15 (10.9 %)
1 15 (10.9 %) 9 (6.6 %)
2 14 (10.2 %) 3 (2.2 %)
3 1 (0.7 %) 0 (0.0 %)
Grading (G) 1 5 (3.6 %) 0 (0.0 %)
2 41 (29.9 %) 16 (11.6 %)
3 44 (32.1 %) 17 (12.4 %)
Mean age at surgery 64,2 64,56
(median age) (65) (67)
Sex (m:w) 68:34 30:5
Median OS (months) 52 24
(SD; 95 % CI [months]) (±23.7; 5.5– 98.5) (± 12.8;0.0– 49.0)

 

Immunohistochemical detection of CD24 expression using clone SWA11 and SN3b

Using the mAb SWA11, 47 of 137 (34.3 %) NSCLC revealed CD24 expression (either cytoplasmic or membranous) (Table 2). CD24 expression was observed more frequently in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). In AC cytoplasmic expression was observed more frequently than membranous expression. In SCC, both cyptoplasmic and membranous expression was rare. Normal lung parenchyma (i.e. alveolar surface cells) showed no expression of CD24. Bronchial epithelium showed a strong membranous and cytoplasmic staining of the brush border (Fig. 1).

Table 2

Cytoplasmic and membranous expression of CD24

SWA11 (mAb clone) SN3b (mAB clone)
  AC SCC   AC SCC
Cytoplasmic N (%) N (%) Cytoplasmic N (%) N (%)
0 45 (32.6 %) 19 (13.8 %) 0 76 (55.1 %) 31 (22.5 %)
1 22 (15.9 %) 8 (5.8 %) 1 12 (8.7 %) 1 (0.7 %)
2 17 (12.3 %) 4 (2.9 %) 2 7 (5.1 %) 2 (1.4 %)
3 18 (13.0 %) 4 (2.9 %) 3 1 (0.7 %) 0 (0 %)
AC SCC AC SCC
Membranous N (%) N (%) Membranous N (%) N (%)
0 68 (49.3 %) 21 (15.2 %) 0 64 (46.4 %) 30 (21.7 %)
1 21 (15.2 %) 5 (3.6 %) 1 10 (7.2 %) 2 (1.4 %)
2 8 (5.8 %) 4 (2.9 %) 2 12 (8.7 %) 2 1.4 %)
3 5 (3.6 %) 5 (3.6 %) 3 10 (7.2 %) 0 (0 %)

Staining intensities are determined as follows:

0: negative or equivocal, 1: weak, 2: moderate and 3: strong CD24 staining

 

https://static-content.springer.com/image/art%3A10.1186%2Fs12907-015-0019-z/MediaObjects/12907_2015_19_Fig1_HTML.gif

Fig 1

The immunohistochemical characterization reveals membranous and/or cytoplasmic CD24 (mAb SWA11) expression. Strong cytoplasmic CD24 expression is found in a proportion of both AC (a) and SCC (b, d) specimens. Membranous CD24 expression can be pronounced with only scant or even absent cytoplasmic staining as shown in the AC (c). Also, both membranous and cytoplasmic CD24 detection can be found in some instances (d), the insert is showing the corresponding squamous carcinoma in-situ with membranous staining. Simultaneous membranous and cytoplasmic CD24 expression is also found in AC specimens (e, f). In normal tissue, alveolar epithelial cells do not express CD24 (g), whereas CD24 staining is found at the apical cell membrane of bronchial respiratory epithelia (h)

Using the mAb SN3b, 29 of 137 (21.2 %) NSCLC revealed CD24 expression (either cytoplasmic or membranous) (Table 2). As above, CD24 expression was observed more frequently in adenocarcinomas (AC) than in squamous cell carcinomas (SCC). However, in contrast to mAb SWA11 cytoplasmic expression was observed less frequently than membranous expression in AC. In SCC, both cytoplasmic and membranous expression was rare. Normal lung parenchyma (i.e. alveolar surface cells) showed a distinct membranous immunoreactivity. Bronchial epithelium revealed both membranous and cytoplasmic staining of CD24.

Correlation between SWA11 and SN3b: As SWA11 and SN3b detect different epitopes, we evaluated the correlation of the immunohistochemical staining patterns. Of 132 NSCLC specimens with matched expression data, only 9 specimens (6.8 %) revealed a concordant CD24 expression. Of these cases, 4 cases revealed a concordant cytoplasmic staining and another 5 cases revealed a concordant membranous CD24 expression. Statistically, no significant correlation between the two mAb could be observed (cc = −0.63, p = 0.470; Fisher’s exact test p = 0.665). The correlation of cytoplasmic and membranous expression (for each antibody) was as follows: cc = 0.475 (p < 0.05) for SWA11 (n = 108) and cc = 0.140 (p = 0.11) for SN3b (n = 103).

Survival analyses

Recent studies indicate that CD24 expression is associated with tumor progression and poorer survival rates. Therefore, we performed follow up analyses with a special emphasis on 1) the prognostic value of mAb SWA11 in dependence on subcellular staining characteristics and 2) the prognostic values of different clinicopathological parameters:

Prognostic value of CD24 in Kaplan Meier Analyses

Only membranous CD24 (SWA11) staining revealed significantly poorer survival rates (median overall survival 21 vs. 52 months; p = 0.005) as illustrated in Fig. 2. In contrast, cytoplasmic CD24 (SWA11) staining did not affect the survival rates (median OS 34 vs. 35 months; p = 0.884) (Table 3). When stratifying the cohort into SCC (n = 35) and AC (n = 102) in Kaplan Meier analyses, membranous CD24 (SWA11) expression did not affect patients’ survival, neither in SCC (p = 0.243) nor AC (p = 0.135) (Table 3), probably due to the small number of observations (Fisher exact test: p > 0.05). After stratification for AC subtypes, membranous CD24 expression (SWA11) showed a tendency towards an association with poorer survival in acinar subtype AC, but failed significance (p = 0.328).
https://static-content.springer.com/image/art%3A10.1186%2Fs12907-015-0019-z/MediaObjects/12907_2015_19_Fig2_HTML.gif

Fig 2

Survival analysis. Kaplan-Meier curves according to SWA11 expression. Cases with moderate to strong expression were bundled in a ‘high expression’ and cases with negative or weak expression in a ‘low expression’ group. Membranous expression of CD24 detected by SWA11 proved to be an independent marker for shorter survival times in NSCLC (p = 0.005)

Table 3

Univariate survival analysis

SWA11 No. of cases Mean survival time Median survival time p-value
(months +/− s.e.) (months +/− s.e.)
Mem CD24
Negative 76 84.833 +/− 10.395 52.000 +/− 27.030 0.005
Positive 16 27.925 +/− 6.379 21.000 +/− 4.000
Cyto CD24
Negative 66 75.209 +/− 10.577 35.000 +/− 12.422 0.884
Positive 26 60.540 +/− 11.551 34.000 +/− 12.196
Total CD24
Negative 64 76.972 +/− 10.841 35.000 +/− 13.726 0.633
Positive 28 57.535 +/− 10.895 34.000 +/− 9.303
SCC
Mem CD24 negative 16 52.063 +/− 14.668 16.000 +/− 16.000 0.243
Mem CD24 positive 7 21.571 +/− 7.201 24.000 +/− 23.568
AC
Mem CD24 negative 59 88.953 +/− 11.631 56.000 +/− 22.885 0.135
Mem CD24 positive 8 39.167 +/− 11.674 21.000 +/− 8.485
pN0 31 103.641 +/− 14.940 93.000 +/− 28.224 0.012
pN1+ 30 54.911 +/− 10.646 26.000 +/− 0.983

 

…..

Univariate survival analysis according to the Cox regression model (mAb SWA11)

  Beta HR (hazard ratio) 95 % CI of HR P-value
SWA11 mem all 0.856 2.353 1.268–4.364 0.007
pN 0.963 2.620 1.389–4.943 0.003
pT 0.844 2.325 1.279–4.224 0.006
Tumour type 0.975 2.651 1.999–3.517 0.000

Table 5

Multivariate survival analysis according to the Cox regression model (mAb SWA11)

  Beta HR (hazard ratio) 95 % CI of HR P-value
SWA11 mem all 0.944 2.571 1.211–5.458 0.014
pN 0.737 2.091 1.087–4.021 0.027
pT 0.587 1.799 0.755–4.283 0.185

 

…..

In the present study, we have analyzed immunohistochemical staining characteristics and the prognostic value of CD24 expression in NSCLC with a special emphasis on the comparison of the CD24 antibodies SWA11 and SN3b. The most important result of our study is that the prognostic relevance of CD24 is critically dependent on the careful consideration of sub-cellular compartments and the epitope specificity of the antibody used.

Overall, about one third of the NSCLC cohort revealed a significant CD24 expression (either cytoplasmic or membranous). These results are in line with the findings of other studies. In another NSCLC cohort, CD24 (SN3b) expression was found in 33 % of the samples (87 of 267 cases) [2]. Consistent with those results, we have found similar rates of high CD24 expression levels (35 % of the cases) for SWA11. Originally, we would have expected lower rates than those found by Lee et al, as they used the antibody SN3b, that also recognizes yet unidentified other glycoproteins next to CD24. Furthermore, they used whole mount sections instead of tissue microarrays. A possible explanation for rather equal detection rates would be the fact that it has been demonstrated that the epitope recognized by SN3b is indeed present in CD24, but is not found in all glycoforms of CD24 [14]. In contrast to the commonly used mAb SN3b, mAb SWA11 binds to the protein core of CD24 and does not depict other glycan moieties next to CD24. The protein core of CD24 is linear, consisting of the amino acid sequence leucine-proline-alanine (LAP) next to a glycosyl-phosphatidylinositol anchor [15].

CD24 expression has been associated with disease progression and cancer-related death in the majority of malignant tumors [2, 3, 16, 17], although a caveat to these data is that most of these studies are based on the supposedly less specific CD24 clone SN3b. Lee et al demonstrated a significant association between CD24-high expression (SN3b) and shorter patient survival times. Furthermore, Lee and colleagues and ourselves in former studies referred the results to cytoplasmic CD24 expression [2, 3].

Switching Off Cancers’ Ability to Spread

http://www.technologynetworks.com/rnai/news.aspx?ID=189704

A key molecule in breast and lung cancer cells can help switch off the cancers’ ability to spread around the body.

The findings by researchers at Imperial College London, published in the journal EMBO Reports, may help scientists develop treatments that prevent cancer travelling around the body – or produce some kind of test that allows doctors to gauge how likely a cancer is to spread. During tumour growth, cancer cells can break off and travel in the bloodstream or lymph system to other parts of the body, in a process called metastasis.

Patients whose cancers spread tend to have a worse prognosis, explains Professor Justin Stebbing, senior author of the study from the Department of Surgery and Cancer at Imperial: “The ability of a cancer to spread around the body has a large impact on a patient’s survival. However, at the moment we are still in the dark about why some cancers spread around the body – while others stay in one place. This study has given important insights into this process.”

The researchers were looking at breast and lung cancer cells and they found that a protein called MARK4 enables the cells to break free and move around to other parts of the body, such as the brain and liver. Although scientist are still unsure how it does this, one theory is it affects the cell’s internal scaffolding, enabling it to move more easily around the body. The team found that a molecule called miR-515-5p helps to silence, or switch off, the gene that produces MARK4.

In the study, the team used human breast cancer and lung cancer cells to show that the miR-515-5p molecule silences the gene MARK4. They then confirmed this in mouse models, which showed that increasing the amount of miR-515-5p prevents the spread of cancer cells. The findings also revealed that the silencer molecule was found in lower levels in human tumours that had spread around the body. The team then also established that patients with breast and lung cancers whose tumours had low amounts of these silencer molecules – or high amounts of MARK4 – had lower survival rates.

Researchers are now investigating whether either the MARK4 gene or the silencer molecule could be targeted with drugs. They are also investigating whether these molecules could be used to develop a test to indicate whether a patient’s cancer is likely to spread. Professor Stebbing said: “In our work we have shown that this silencer molecule is important in the spread of cancer. This is very early stage research, so we now need more studies to find out more about this molecule, and if it is present in other types of cancer.”

Dr Olivier Pardo, lead author of the paper, also from the Department of Surgery and Cancer at Imperial, added: “Our work also identified that MARK4 enables breast and lung cancer cells to both divide and invade other parts of the body. These findings could have profound implications for treating breast and lung cancers, two of the biggest cancer killers worldwide.” The study was supported by the NIHR Imperial Biomedical Research Centre, the Medical Research Council, Action Against Cancer and the Cancer Treatment and Research Trust.

 

‘Silencer molecules’ switch off cancer’s ability to spread around body

by Kate Wighton

main image

Scientists have revealed that a key molecule in breast and lung cancer cells can help switch off the cancers’ ability to spread around the body

The findings by researchers at Imperial College London, published in the journal EMBO Reports, may help scientists develop treatments that prevent cancer travelling around the body – or produce some kind of test that allows doctors to gauge how likely a cancer is to spread.

During tumour growth, cancer cells can break off and travel in the bloodstream or lymph system to other parts of the body, in a process called metastasis.

Patients whose cancers spread tend to have a worse prognosis, explains Professor Justin Stebbing, senior author of the study from the Department of Surgery and Cancer at Imperial: “The ability of a cancer to spread around the body has a large impact on a patient’s survival. However, at the moment we are still in the dark about why some cancers spread around the body – while others stay in one place. This study has given important insights into this process.”

The researchers were looking at breast and lung cancer cells and they found that a protein called MARK4 enables the cells to break free and move around to other parts of the body, such as the brain and liver. Although scientist are still unsure how it does this, one theory is it affects the cell’s internal scaffolding, enabling it to move more easily around the body.

 

miR‐515‐5p controls cancer cell migration through MARK4 regulation

Olivier E Pardo, Leandro Castellano, Catriona E Munro, Yili Hu, Francesco Mauri,Jonathan Krell, Romain Lara, Filipa G Pinho, Thameenah Choudhury, Adam EFrampton, Loredana Pellegrino, Dmitry Pshezhetskiy, Yulan Wang, JonathanWaxman, Michael J Seckl, Justin Stebbing    

EMBO reports http://embor.embopress.org/content/early/2016/02/10/embr.201540970     http://dx.doi.org:/
Here, we show that miR‐515‐5p inhibits cancer cell migration and metastasis. RNA‐seq analyses of both oestrogen receptor receptor‐positive and receptor‐negative breast cancer cells overexpressing miR‐515‐5p reveal down‐regulation of NRAS, FZD4, CDC42BPA, PIK3C2B and MARK4 mRNAs. We demonstrate that miR‐515‐5p inhibits MARK4 directly 3′ UTR interaction and that MARK4 knock‐down mimics the effect of miR‐515‐5p on breast and lung cancer cell migration. MARK4 overexpression rescues the inhibitory effects of miR‐515‐5p, suggesting miR‐515‐5p mediates this process through MARK4 down‐regulation. Furthermore, miR‐515‐5p expression is reduced in metastases compared to primary tumours derived from both in vivo xenografts and samples from patients with breast cancer. Conversely, miR‐515‐5p overexpression prevents tumour cell dissemination in a mouse metastatic model. Moreover, high miR‐515‐5p and low MARK4 expression correlate with increased breast and lung cancer patients’ survival, respectively. Taken together, these data demonstrate the importance of miR‐515‐5p/MARK4 regulation in cell migration and metastasis across two common cancers.
Embedded Image

miR‐515‐5p inhibits cancer progression, cell migration and metastasis through its direct target MARK4, a regulator of the cytoskeleton and cell motility. Moreover, reduced miR‐515‐5p and increased MARK4 levels in metastatic lung and breast cancer correlate with poor patient prognosis.

  • MARK4 down‐regulation promotes microtubule polymerisation.

  • Increased cell spreading downstream of miR‐515‐5p overexpression or MARK4 silencing hinders cell motility and invasiveness.

  • miR‐515‐5p overexpression or MARK4 silencing prevent organ colonisation by circulating tumour cells.

  • MARK4 inhibitors may represent novel therapeutic agents to control cancer dissemination.breasat cancer

 

Liquid Biopsy for NSCLC

http://www.technologynetworks.com/Diagnostics/news.aspx?ID=190276

‘Liquid biopsy’ blood test accurately detects key genetic mutations in most common form of lung cancer, study finds.

A simple blood test can rapidly and accurately detect mutations in two key genes in non-small cell lung tumors, researchers at Dana-Farber Cancer Institute and other institutions report in a new study – demonstrating the test’s potential as a clinical tool for identifying patients who can benefit from drugs targeting those mutations.

The test, known as a liquid biopsy, proved so reliable in the study that Dana-Farber/Brigham and Women’s Cancer Center (DF/BWCC) expects to offer it soon to all patients with non-small cell lung cancer (NSCLC), either at the time of first diagnosis or of relapse following previous treatment.

NSCLC is the most common form of lung cancer, diagnosed in more than 200,000 people in the United States each year, according to the American Cancer Society. An estimated 30 percent of NSCLC patients have mutations in either of the genes included in the study, and can often be treated with targeted therapies. The study is being published online today by the journal JAMA Oncology.

The liquid biopsy tested in the study – technically known as rapid plasma genotyping – involves taking a test tube-full of blood, which contains free-floating DNA from cancer cells, and analyzing that DNA for mutations or other abnormalities. (When tumor cells die, their DNA spills into the bloodstream, where it’s known as cell-free DNA.) The technique, which provides a “snapshot” of key genetic irregularities in a tumor, is a common tool in research for probing the molecular make-up of different kinds of cancers.

“We see plasma genotyping as having enormous potential as a clinical test, or assay – a rapid, noninvasive way of screening a cancer for common genetic fingerprints, while avoiding the challenges of traditional invasive biopsies,” said the senior author of the study, Geoffrey Oxnard, MD, thoracic oncologist and lung cancer researcher at Dana-Farber and Brigham and Women’s Hospital. “Our study was the first to demonstrate prospectively that a liquid biopsy technique can be a practical tool for making treatment decisions in cancer patients. The trial was such a success that we are transitioning the assay into a clinical test for lung cancer patients at DF/BWCC.”

The study involved 180 patients with NSCLC, 120 of whom were newly diagnosed, and 60 of whom had become resistant to a previous treatment, allowing the disease to recur. Participants’ cell-free DNA was tested for mutations in the EGFR and KRAS genes, and for a separate mutation in EGFR that allows tumor cells to become resistant to front-line targeted drugs. The test was performed with a technique known as droplet digital polymerase chain reaction (ddPCR), which counts the individual letters of the genetic code in cell-free DNA to determine if specific mutations are present. Each participant also underwent a conventional tissue biopsy to test for the same mutations. The results of the liquid biopsies were then compared to those of the tissue biopsies.

The data showed that liquid biopsies returned results much more quickly. The median turnaround time for liquid biopsies was three days, compared to 12 days for tissue biopsies in newly diagnosed patients and 27 days in drug-resistant patients.

Liquid biopsy was also found to be highly accurate. In newly diagnosed patients, the “predictive value” of plasma ddPCR was 100 percent for the primary EGFR mutation and the KRAS mutation – meaning that a patient who tested positive for either mutation was certain to have that mutation in his or her tumor. For patients with the EGFR resistance mutation, the predictive value of the ddPCR test was 79 percent, suggesting the blood test was able to find additional cases with the mutation that were missed using standard biopsies.

“In some patients with the EGFR resistance mutation, ddPCR detected mutations missed by standard tissue biopsy,” Oxnard remarked. “A resistant tumor is inherently made up of multiple subsets of cells, some of which carry different patterns of genetic mutations. A single biopsy is only analyzing a single part of the tumor, and may miss a mutation present elsewhere in the body. A liquid biopsy, in contrast, may better reflect the distribution of mutations in the tumor as a whole.”

When ddPCR failed to detect these mutations, the cause was less clear-cut, Oxnard says. It could indicate that the tumor cells don’t carry the mutations or, alternatively, that the tumor isn’t shedding its DNA into the bloodstream. This discrepancy between the test results and the presence of mutations was less common in patients whose cancer had metastasized to multiple sites in the body, researchers found.

The ddPCR-based test, or assay, was piloted and optimized for patients at the Translational Resarch lab of the Belfer Center for Applied Cancer Science at Dana-Farber. It was then validated for clinical use at Dana-Farber’s Lowe Center for Thoracic Oncology.

An advantage of this form of liquid biopsy is that it can help doctors quickly determine whether a patient is responding to therapy. Fifty participants in the study had repeat testing done after starting treatment for their cancer. “Those whose blood tests showed a disappearance of the mutations within two weeks were more likely to stay on the treatment than patients who didn’t see such a reduction,” said the study’s lead author, Adrian Sacher, MD, of Dana-Farber and Brigham and Women’s Hospital.

And because tumors are constantly evolving and acquiring additional mutations, repeated liquid biopsies can provide early detection of a new mutation – such as the EGFR resistance mutation – that can potentially be treated with targeted agents.

“The study data are compelling,” said DF/BWCC pathologist Lynette Sholl, MD, explaining the center’s decision to begin offering ddPCR-based liquid biopsy to all lung cancer patients. “We validated the authors’ findings by cross-comparing results from liquid and tissue biopsies in 34 NSCLC patients. To work as a real-world clinical test, liquid biopsy needs to provide reliable, accurate data and be logistically practical. That’s what we’ve seen with the ddPCR-based blood test.

“The test has great utility both for patients newly diagnosed with NSCLC and for those with a recurrence of the disease,” she continued. “It’s fast, it’s quantitative (it indicates the amount of mutant DNA in a sample), and it can be readily employed at a cancer treatment center.”

The co-authors of the study are Cloud Paweletz, PhD, Allison O’Connell, BSc, and Nora Feeney, BSc, of the Belfer Center for Applied Cancer Science at Dana-Farber; Ryan S. Alden BSc, and Stacy L. Mach BA, of Dana-Farber; Suzanne E. Dahlberg, PhD, of Dana-Farber and Harvard T.H. Chan School of Public Health; and Pasi A. Jänne, MD, PhD, of Dana-Farber, the Belfer Center, and Brigham and Women’s Hospital.

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IsomicroRNA

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

GEN Feb 15, 2016 (Vol. 36, No. 4)

MicroRNAs Rise from Trash to Treasure  

MicroRNAs Are More Plentiful and More Subtle In Action Than Was Once Suspected

Richard A. Stein, M.D., Ph.D.

 

One of the unexpected findings of the Human Genome Project was that over 98% of the human genome does not encode for proteins. Once dismissed as “junk” genomic material, non-protein-coding DNA is now appraised more highly.

Or to be more precise, at least some portions of non-protein-coding DNA are thought to serve important biological functions.

For example, some stretches of DNA give rise to a noncoding but still functional kind of RNA called microRNA. MicroRNAs have increasingly emerged in recent years as key regulators of biological processes and pathways.

During the years since their discovery, a key question in the biology of microRNAs has focused on the number of microRNAs encoded in the genome. Between 1993 and 2015, approximately 1,900 human genome loci were discovered to produce microRNAs and were added to miRBbase, the public database that catalogues and annotates microRNA molecules.

The cataloguing of microRNAs work has been pursued with extra urgency since 2004, the year the connection between microRNAs and human disease was first demonstrated. “When this connection was made, it launched a whole new field,” says Isidore Rigoutsos, Ph.D., professor of pathology, anatomy, and cell biology and director of the Computational Medicine Center at Thomas Jefferson University.

 

 

 

Another Set of MicroRNAs Emerge

“We wanted to know how many microRNA-producing loci really exist in humans,” recalls Dr. Rigoutsos. In a study published in 2015, Dr. Rigoutsos and colleagues analyzed datasets from 1,323 individuals that represented 13 different tissues and identified an additional 3,356 such genomic loci that produce (at least) 3,707 novel microRNs

“We basically tripled the number of locations in the human genome that are now known to encode microRNAs,” asserts Dr. Rigoutsos. Considering that each microRNA regulates up to hundreds of different mRNAs, and that each mRNA is regulated by tens of microRNAs, this finding adds a new layer of complexity to the regulatory dynamics of the human transcriptome.

The newly unveiled microRNAs and previously characterized microRNAs have distinct expression patterns. While 50–60% of the microRNAs previously deposited into the miRBase are expressed in multiple tissues, only about 10% of the newly discovered microRNAs are shared across multiple tissue types. Also, most of the newly found microRNAs show tissue-specific expression.

Using Argonaute CLIP-seq data, Dr. Rigoutsos and colleagues showed that similar percentages of the two sets of microRNAs were in complex with Argonaute proteins. “This shows that these novel microRNAs participate in RNA interference just as frequently as the miRBase microRNAs,” contends Dr. Rigoutsos.

In a comparative analysis between the human microRNA datasets and the chimpanzee, gorilla, orangutan, macaque, mouse, fruit fly, and mouse genomes, Dr. Rigoutsos and colleagues discovered that almost 95% of the newly unveiled microRNAs were primate-specific, and over 56% of them were found only in humans.

“We are seeing many human microRNAs that do not exist in the mouse,” states Dr. Rigoutsos. “This means that the mouse models engineered to capture human disease cannot recapitulate the interactions mediated by these microRNAs.

 

  • Interest in IsomiRs Grows

  • In the years since the biology of microRNAs started receiving increasing attention, the conventional view has been that one microRNA locus generates one microRNA. However, once deep sequencing became widely available, microRNA variants that showed differences at their 5′- or 3′-termini have been described.

    “It was initially presumed that these variants were likely the result of the enzyme Dicer not being sufficiently accurate when processing microRNA precursors,” notes Dr. Rigoutsos. Subsequent research revealed that microRNAs are more dynamic than previously thought, with each precursor being able to generate multiple mature microRNA species known as isomiRs.

    To gain insight into the biology of isomiRs, Dr. Rigoutsos and colleagues analyzed genomic datasets from 452 individuals participating in the 1000 Genomes Project. The datasets comprised five different populations and two races. In addition, each population was represented by an even number of men and women.

    This collection allowed the abundance of microRNA isoforms to be examined with respect to population, gender, and race. “We found that isomiRs have expression profiles that are population-, race-, and gender-dependent,” informs Dr. Rigoutsos.

    All the transcriptome data that this analysis was based on came from immortalized B cells. “These are cells that normally are not associated with gender differences, but molecularly we found, in these cells, differences between men and women of the same population and race,” explains Dr. Rigoutsos.

  • Expanding these observations to disease states, Dr. Rigoutsos and colleagues collected isomiR profiles from tissue affected by breast cancer, and compared them with isomiR profiles from control breast tissue. The investigators found that the isomiR profiles also depend on tissue state (healthy vs. diseased), on disease subtype, and on the patient’s race.

    For example, their analysis identified several miR-183-5p isoforms that were upregulated in white triple-negative breast cancer patients compared to control breast samples, but not in black/African-American triple-negative breast cancer patients. In an in vitro phase of this study, three isoforms of this microRNA species were overexpressed in human breast cancer cell lines.

    “We found very little overlap in the gene sets that were affected by each of these isoforms,” emphasizes Dr. Rigoutsos. Despite being generated simultaneously by the same locus, each of the three isoforms affected distinct groups of genes, thus exerting different effects on the transcriptome.

    “As the relative abundance of these isoforms changes ever so slightly from patient to patient, it will affect the corresponding gene groups slightly differently,” concludes Dr. Rigoutsos. “In the process, it creates a new molecular background in each patient.”

    MicroRNAs Point to Therapeutic Strategies against Colorectal Cancer

  • “We are using microRNAs as modulators to overcome chemotherapy resistance in colorectal cancer,” says Jingfang Ju, Ph.D., associate professor of pathology and co-director of translational research at Stony Brook University School of Medicine. Resistance to chemotherapy is one of the major challenges in the clinical management of malignancies, including colorectal cancer. Chemotherapy is usually unable to eliminate cancer stem cells, which may become even more resistant over time, and several microRNAs have been implicated in this process.  “We reasoned that we could provide new modulatory approaches to target this small cell population and allow chemotherapy, radiotherapy, or immunotherapy to eliminate resistant populations or at least prolong long-term survival,”  Dr. Ju said.
  • http://www.genengnews.com/Media/images/Article/StonyBrookUniv_JingfangJu5310853233.jpg

    This image shows how miR-129 may function as a tumor suppressor in colorectal cancer. In this model, which has been proposed by researchers at Stony Brook University’s Translational Research Laboratory, miR-129 suppresses the protein expression of three critical targets—BCL2, TS, and E2F3. Downregulation of BCL2 activates the intrinsic apoptosis pathway by cleaving caspase-9 and caspase-3. Downregulation of TS and E2F3 inhibits cell proliferation by impacting the cell cycle. Consequently, miR-129 exerts a strong antitumor phenotype by induction of apoptosis and impairment of proliferation in tumor cells. [Mihriban Karaayvaz, Haiyan Zhai, Jingfang Ju]

     

    In a retrospective study in which colorectal patient samples were used, Dr. Ju and colleagues revealed that hsa-miR-140-5p expression progressively decreases from normal tissues to primary colorectal cancer tissue, and that it shows a further decrease in liver and lymph node metastases. The experimental overexpression of hsa-miR-140-5p inhibited colorectal cancer stem cell growth by disrupting autophagy, and in a mouse model of disease it abolished tumor formation and metastasis.

    In addition to hsa-miR-140-5p, Dr. Ju and colleagues recently identified hsa-miR-129 and found that it, too, has therapeutic potential. Specifically, they showed that hsa-miR-129 enhanced the sensitivity of colorectal cancer cells to 5-fluorouracil, pointing toward its ability to function as a tumor suppressor.

    One of the mechanisms implicated in this process was the ability of miR-192 to inhibit protein translation of several important targets. These include Bcl-2 (B-cell lymphoma 2), a key anti-apoptotic protein; E2F3, a major cell cycle regulator; and thymidylate synthase, an enzyme that is inhibited by 5-fluorouracil.

    The NIH recently awarded a $3 million grant to establish the Long Island Bioscience Hub (LIBH), which is part of the NIH’s Research Evaluation and Commercialization Hub (REACH) program and represents a partnership between the Center for Biotechnology, Stony Brook University, Cold Spring Harbor Laboratory, and Brookhaven National Laboratory. One of the technology development grants, as part of the first funding cycle of this initiative, will support a feasibility investigation of hsa-miR-129-based therapeutics in colon cancer, an effort led by Dr. Ju. “We are further exploring this novel mechanism,” states Dr. Ju. “We anticipate conducting pharmacokinetic studies and moving to a clinical trial in the future.”

    MicroRNA Insights Gleaned from Host-Virus Interactions

    http://www.genengnews.com/Media/images/Article/MtSinaiHosp_Benjamin_tenOever1664523413.jpg

    At Mount Sinai Hospital’s Icahn School of Medicine, researchers used a codon-optimized version of VP55 produced from an adenovirus-based vector to study the impact of microRNA deletion on the response to virus infection. This image shows RNA in situ hybridization of fibroblasts expressing VP55 (top left), and that of mock-treated fibroblasts (bottom right). Ribosomal RNA, DNA, and microRNAs (miR-26) are depicted by red, blue (DAPI), and green fluorophores, respectively.

    “We observed that when a poxvirus is artificially engineered to encode a microRNA, the microRNA is destroyed along with all the microRNAs from the host cell,” says Benjamin R. tenOever, Ph.D., professor of microbiology at the Icahn School of Medicine, Mount Sinai Hospital. Previously, Dr. tenOever’s group reported that a single vaccinia virus-encoded gene product, VP55, is sufficient to achieve this effect. The group also found that the protein adds nontemplate adenosines to the 3′-end of microRNAs associated with the RNA-induced silencing complex.

    biology,” asserts Dr. tenOever.

    In a recent study, Dr. tenOever and colleagues used a codon-optimized version of VP55 produced from an adenovirus-based vector to study the impact microRNA deletion would have on our normal response to virus infection. “We found that after administration of the vector and rapid ablation of microRNA expression, there is very little that happens over the first one to two days,” informs Dr. tenOever. During the first 24–48 hours after VP55 delivery, the elimination of cellular microRNAs impacted less than 0.35% of the over 11,000 genes expressed in the cell. After 9 days, however, almost 20% of the genes showed significant changes in expression.

    “MicroRNAs are very powerful and influential in controlling the biology of the cell but they do so over the long term,” declares Dr. tenOever. These findings are in agreement with knowledge that has accumulated over the years about microRNA biology, which established that microRNAs play a central role in determining how cells differentiate during development.

    “While microRNAs can act on hundreds of mRNAs, their action requires several days of fine-tuning to have long-term consequences,” adds Dr. tenOever. This finding suggests miRNAs are unable to significantly contribute to the acute response to virus infection.

    The one exception to this observation was that, even though very few genes were affected in the first 48 hours after VP55 delivery, several genes encoding chemokines were impacted. These included chemokines responsible for recruiting antigen-presenting cells, neutrophils, and other immune cells.

    An in vivo analysis of mouse lung tissue 48 hours after vector administration confirmed that several cytokines were specifically upregulated, resulting in immune cell infiltration following the degradation of all microRNAs. These results indicate that the acute viral infection is largely independent of microRNAs, and that microRNAs are primarily involved in the adaptive response to infection and other longer term processes.

    • MicroRNA Biomarkers Reveal Molecular Pathways of Kidney Damage

      “Our approach involves looking at microRNAs from the perspective of biomarkers as a readout for kidney damage,” says Vishal S. Vaidya, Ph.D., associate professor of medicine and environmental health at Brigham and Women’s Hospital, Harvard Medical School, and Harvard T.H. Chan School of Public Health. “At the same time, we are exploring their utility as therapeutics.”

      A large number of medications and occupational toxins cause kidney damage, but many tests to assess kidney function and damage are not sufficiently sensitive or specific, opening the need for novel diagnostic strategies. MicroRNAs, which are differentially expressed between healthy and diseased states, are promising as early biomarkers for impaired renal function.

      “MicroRNAs can also provide information about which pathways are active and which targets can be druggable,” points out Dr. Vaidya.

      In a study that used microRNAs and proteins to provide a combined biomarker signature, Dr. Vaidya and colleagues examined two patient cohorts, one presenting with acetaminophen-induced kidney injury and the other one with cisplatin-induced kidney damage. “Protein biomarkers provide sensitivity, and microRNAs offer mechanistic insight,” explains Dr. Vaidya.

      This approach helped visualize metabolic pathways that are altered in the kidney during toxic injury. “The biggest challenge, from a therapeutic perspective, is that microRNAs regulate many mRNAs and, therefore, impact many proteins,” concludes Dr. Vaidya.

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Blocking miRNAs in Triple Negative Breast Cancer

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Why Blocking microRNAs in Triple Negative Breast Cancer Is so Difficult

http://www.genengnews.com/gen-news-highlights/why-blocking-micrornas-in-triple-negative-breast-cancer-is-so-difficult/81252048/

New research uncovers why attempts at blocking microRNAs for triple negative breast cancer often fail. [National Breast Cancer Foundation]

http://www.genengnews.com/Media/images/GENHighlight/thumb_Dec2_2015_NationalBreastCancerFoundation_MicroRNAs4198253108.jpg

 

While a triple negative score is hardly ever a good thing, for breast cancer it is especially troubling. Triple-negative breast cancer (TNBC) refers to a disease scenario where the cancer cells do not express the genes for estrogen, progesterone, or HER2/neu receptors simultaneously—making this cancer particularly aggressive and difficult to treat as most chemotherapies target one of these receptors.

Over the past several years, researchers have discovered that various microRNAs (miRNAs) underlie the expression of certain genes that can enable cancer cells to proliferate faster. However, the ability to block these miRNAs in TNBC has been met with failure. Yet now, scientists from Thomas Jefferson University in Philadelphia believe they have discovered the reason conventional methods to block these miRNAs has been thus far unsuccessful.

“Triple-negative breast cancer is one of the most aggressive forms of breast cancer, and there’s been a lot of excitement in blocking the microRNAs that appear to make this type of cancer grow faster and resist conventional treatment,” explained senior author Eric Wickstrom, Ph.D., professor in the department of biochemistry and molecular biology at TJU. “However blocking microRNAs hasn’t met with great success and this paper offers one explanation for why that might be the case.”

The findings from this study were published online today in PLOS ONE through an article entitled “Non-specific blocking of miR-17-5p guide strand in triple negative breast cancer cells by amplifying passenger strand activity.” Insight from this study may enable new and more effective design of blockers against previously intractable miRNAs.

“Triple negative breast cancer strikes younger women, tragically killing them in as little as two years,” noted lead author Yuan-Yuan Jin, a doctoral candidate in the department of biochemistry and molecular biology at TJU. “Only chemotherapy and radiation are approved therapies for triple negative breast cancer. We want to treat a genetic target that will keep patients alive with a good quality of life.”

The investigators targeted the miRNA molecule miR-17, which has been shown previously to cause a surge in TNBC growth by alternating genes that would normally signal a diseased or early cancerous cell to die—specifically, the tumor suppressor genes PDCD4 and PTEN.

Although, when the TJU researchers tried to reduce the levels of miR-17 in TNBC cells, rather than increase the levels of the tumor suppressor genes, as they had anticipated, they saw an even larger decrease in these genes than unmodified controls.

The TJU team was acting under the current assumptions that miRNA, which are double stranded, only silence genes using one of their two strands, which is complementary to parts of the messenger RNA coding sequence. The matching, or so-called passenger strand, was thought to be discarded and degraded by the cell.

Using a method to silence RNAs, which involves flooding the cell with modified RNA sequences that mimic the passenger strand and bind to the single-stranded microRNA before it reaches its target, the TJU team saw more silencing of the PDCD4 and PTEN genes.  After some bioinformatic and folding energy calculations, the authors realized that both strands of miR-17 were active in downregulating the tumor suppressor genes.

“Rather than blocking miR-17, we were inadvertently boosting its levels, and, therefore, boosting the cell’s cancerous potential,” noted Jin.

The results of the current study should help to open a pathway to designing specific blockers of one microRNA strand without imitating the opposite strand. Dr. Wickstrom added that “we are now testing new miR-17 blocker designs made possible by these results.”

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Small but mighty RNAs

Larry H Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E. 2; 3.5

Revised 9/30/2015

Albert Lasker
Basic Medical Research Award

Victor Ambros, David Baulcombe, and Gary Ruvkun

For discoveries that revealed an unanticipated world of tiny RNAs that regulate gene function in plants and animals

The 2008 Albert Lasker Award for Basic Medical Research honors three scientists who discovered an unanticipated world of tiny RNAs that regulate gene activity in plants and animals. Victor R. Ambros (University of Massachusetts Medical School, Worcester) and Gary B. Ruvkun (Massachusetts General Hospital, Boston, Harvard Medical School) unearthed the first example of this type of molecule in animals and demonstrated how the RNAs turn off genes whose activities are crucial for development. David C. Baulcombe (University of Cambridge) established that small RNAs silence genes in plants as well, thus catalyzing discoveries of many such RNAs in a wide range of living things. His findings led to the identification of the biochemical machinery that unifies numerous processes by which small RNAs govern gene activity.

Ambros, Baulcombe, and Ruvkun did not set out to unveil small regulatory RNAs. Ambros and Ruvkun were studying how the worm Caenorhabditis elegans develops from a newly hatched larva into an adult. Baulcombe, in a seemingly unrelated line of inquiry, was probing how plants defend themselves against viruses. All three investigators possessed the open mindedness, wisdom, and experimental finesse to entertain the possibility—and then verify—that tiny RNAs could perform momentous feats. Their work has led to the realization that these molecules are pivotal regulators of normal physiology as well as disease.

RNA—the little molecule that could
In the early 1980s, Ambros joined the laboratory of Robert Horvitz at the Massachusetts Institute of Technology as a postdoctoral fellow. He wanted to outline the means by which genes choreograph the construction of fully formed adults from single cells. Analyses of flies had revealed that certain genes instruct embryos where to place body parts—for example, wings belong on each side and legs belong on the bottom. But Ambros was intrigued by the notion that other genes might specify the timing—rather than the location—of developmental events; alterations in such genes might cause cells and tissues to adopt fates that are normally associated with earlier or later stages of development.

He directed his attention toward one of the first-known genes of this type, called lin-4, which had been identified earlier in the laboratory of Sydney Brenner (Lasker Special Achievement Award, 2000) and subsequently characterized by Horvitz, Martin Chalfie, and John Sulston. Ambros recognized that, during worms’ trek toward adulthood, those with inactive lin-4 get stuck repeating early larval stages. Consequently, they lack cell types and structures typical of fully formed animals and instead contain extra copies of cells ordinarily produced only at early stages. These observations suggested that normal lin-4 allows immature worms to advance past a particular developmental stage; animals with the defective version cannot overcome that hurdle. Ambros discovered that worms lacking a different gene—lin-14—were the antithesis of those with inactive lin-4. The animals skip early steps in development and prematurely acquire characteristics that normally appear later. These and other results suggested that lin-4 and lin-14 exert opposite effects in worm cells.

To dig further into lin-14‘s function and its possible relationship with lin-4, Ruvkun, who by this time (1982) had joined Horvitz’s laboratory as a postdoctoral fellow, collaborated with Ambros to isolate the lin-14 gene. After the investigators set up independent laboratories in the mid 1980s, Ruvkun, at Massachusetts General Hospital in Boston, established that the protein product of lin-14 is abundant during early larval stages and then its quantities plummet. Under conditions in which it unnaturally remains plentiful, early steps repeat, suggesting that the normal drop in the lin-14 protein allows worms to proceed to later stages. Ambros, at Harvard University, found that lin-4 dampens lin-14 activity and thus a picture emerged about how the genes collaborate. At the appropriate time, lin-4 blocks lin-14 and thus allows worms to continue their developmental trajectory.

Ruvkun sought to identify the portion(s) of lin-14 that lin-4 targets, so he tracked down certain genetic anomalies in lin-14‘s sequence that underlie excess production of the lin-14 protein. He found that these alterations reside in the area of the gene that follows the protein blueprint, a span called the 3′ untranslated region (3′ UTR). The perturbations do not influence amounts of the protein’s messenger RNA (mRNA), the molecule that carries genetic information from DNA to the cell’s protein-making factory, Ruvkun showed. Rather, they alter protein quantities. Therefore, molecules that turn off lin-14 after early stages of development presumably exert their effects through the 3′ UTR region of the lin-14 mRNA and prevent the cell from translating its code into protein.

In the meantime, Ambros’s laboratory was isolating the lin-4 gene, which they assumed encoded a protein; although a few RNAs were known to control gene activity in bacteria, conventional wisdom held that, in animal cells, proteins alone enjoy such powers. The team homed in on smaller and smaller pieces of DNA from normal animals that restore typical developmental behavior to a worm that lacks lin-4. Stretches of DNA that were far shorter than standard genes worked. Eventually, the researchers began considering the possibility that its product was an RNA, but they still assumed that the regulatory molecule they sought would be a respectable size. The smallest RNAs known to do anything important in cells contained about 75 nucleotide (nt) building blocks. Eventually, however, their experiments led them to a tiny RNA, composed of about 22 nucleotides. A larger—61 nt—molecule that contained the smaller RNA appeared as well and Ambros noticed that it could fold into a double-stranded “hairpin”—a structure whose significance would become clear years later.

In an exciting exchange of data, Ambros and Ruvkun realized that the 22-nt lin-4 RNA matched sections within the 3′ UTR of the lin-14 mRNA: These sequences could bind one another by the same base-pairing rules that hold together the Watson and Crick DNA strands. In this view, the tiny lin-4 RNA settles on the target lin-14mRNA—in its 3′ UTR—and the resulting double-stranded structure somehow interferes with translation of the lin-14 mRNA’s genetic information into protein (see illustration).

Image of microRNA
Small but mighty.
This scheme shows how one type of tiny RNA, a microRNA (miRNA), silences genes. It is cut out of a precursor hairpin-shaped pre-miRNA to form a mature miRNA, which binds to the 3′ untranslated region (3′ UTR) of a target gene’s messenger RNA and turns off its activity. [Credit: Carin Cain. Based on an illustration from Victor Ambros]

Despite verification that lin-4 was a tiny RNA with huge regulatory powers, these 1993 findings constituted a mere blip on most biologists’ radar screens: lin-4 resided only in worms, so the phenomenon seemed like an oddity that most organisms did not exploit. Worms were exotic in many ways, experts reasoned, and the observation only fueled that attitude.

Branching out to plants and beyond
Across the Atlantic, David Baulcombe, then of the Sainsbury Laboratory in Norwich, UK, was studying how plants resist viruses. When he and others added to viral-infected plants unusual versions of viral genes, the mRNA copies of the normal genes as well as the newly introduced ones disappeared. Similarly, experimentally added non-viral genes suppressed activity of plant genes that contained similar sequences. Baulcombe proposed that such gene silencing occurs when RNAs embrace target mRNA—through typical Watson-Crick base-pairing—and promote destruction of the mRNA or interfere with its translation into protein. However, no one could find such RNAs.

Baulcombe reasoned that the predicted RNAs might have eluded researchers because the molecules were shorter than anyone imagined and thus, experiments had not been designed to detect them. In 1999, he and a postdoctoral fellow in his laboratory, Andrew Hamilton, devised a hunt specifically for small RNAs. They added test genes to plants and found 25-nt long RNAs that matched; furthermore, these small RNAs appeared only under conditions in which target mRNA activity was shut off. The stunning similarity in size between the plant and worm RNAs suggested that small regulatory RNAs exist in many organisms. Furthermore, it hinted at the presence of cellular machinery that dedicates itself to creating these precisely sized molecules and then uses them to quash gene activity.

In 2000, Ruvkun’s laboratory discovered a second tiny regulatory RNA in worms of exactly the same size as thelin-4 RNA and in the same genetic pathway. Similar to the lin-4 RNA, this let-7 RNA dampens activity of its target gene through its 3′ UTR. Furthermore, its sequence too resides within a larger molecule that folds up on itself to form a double-stranded hairpin structure. Later that year, Ruvkun found that many other creatures, including humans, fruit flies, chickens, frogs, zebrafish, mollusks and sea urchins, carry their own versions of let-7, which could also fold into hairpins. The apparent binding site for let-7 RNA in its target was conserved in some of these organisms as well. Moreover, let-7 RNA appeared and disappeared at similar points during development in many of the animals.

The small RNAs, now called microRNAs (miRNAs), had broken through their designation as “worm curiosities.” Researchers realized that the miRNAs likely execute vital functions during growth and development of other creatures as well. Multiple teams raced to expose regulatory RNAs of approximately 22 nucleotides in length. In 2001, Ambros’s group, now at Dartmouth Medical School, in Hanover, as well as those of David Bartel (Massachusetts Institute of Technology) and Thomas Tuschl (Max Planck Institute for Biophysical Chemistry, G�ttingen) discovered almost 100 of these small regulatory RNAs in flies, humans, and worms.

In addition to revealing that small regulatory RNAs dwell in organisms other than worms, Baulcombe’s finding caught many researchers’ attention because it seemed related to a process called RNA interference (RNAi), which had recently exploded onto the biological scene. In RNAi, long RNAs injected into cells hamper gene activity from similar sequences. No one knew why organisms possessed this ability, but presumably it played some role in normal physiology. In 1998, Andrew Fire (Carnegie Institution of Washington, Baltimore) and Craig Mello (University of Massachusetts Medical School, Worcester), published a watershed paper that defined the fundamental features of RNAi (which garnered them the Nobel Prize in 2006). That work yielded the surprising insight that the process depends on double-stranded RNA. However, the means by which double-stranded RNA triggered silencing remained mysterious.

Experiments from Baulcombe’s laboratory provided the crucial clues. Production of the silencing RNA strand depended on the presence of the other strand, he had noticed. This observation suggested that, at some point during manufacture of the small regulatory RNA, it exists as part of a double-stranded molecule. Suddenly it seemed possible that Baulcombe’s tiny RNAs arose by trimming longer molecules of the type that Fire and Mello had discovered. Furthermore, this notion suggested that the hairpin-like lin-4 and let-7 RNAs similarly gave rise to the mature, 22-nt entities.

Scientists wondered whether the cell deployed the same biochemical machinery to create and use RNA molecules that subdued gene activity in all of these gene-silencing systems. However, the mechanisms of the worm miRNAs seemed to differ from those of the plant molecules as well as RNAi. Unlike the system that Ambros and Ruvkun had been untangling, which allowed mRNA to accumulate but thwarted cells’ abilities to translate the information it contained into protein, the plant system and RNAi destroyed mRNA. For that reason and others, many people doubted that the processes were connected. Still the possibility that they shared a common mechanism and machinery tantalized researchers.

In 2001, the Mello, Ruvkun, and Fire groups collaborated to show that efficient liberation of the lin-4 and let-7RNAs from the hairpin molecules relies on the C. elegans version of Dicer, an enzyme that Gregory Hannon (Cold Spring Harbor Laboratory) discovered and named for its ability to chop dsRNA into uniformly sized, small RNAs that direct mRNA destruction during RNAi. These results and others, including similar ones generated by Philip Zamore (University of Massachusetts Medical School, Worcester), cemented the connection between miRNAs and RNAi, thus providing one biological “reason” for the RNAi machinery. Moreover, they identified the apparatus by which cells generate miRNAs and harness them for key pursuits.

Studies in the past several years have indicated that the human genome contains more than 500 and perhaps as many as 1000 miRNAs that could collectively control a third of all of our protein-producing genes. These regulatory molecules have been implicated in a wide range of normal and pathological activities. They play roles not only in embryonic development, but in blood-cell specialization, cancer, muscle function, heart disease, viral infections, and possibly neurological signaling and stem-cell behavior. Researchers are exploring the possibility of using miRNAs “signatures” for diagnosis and prognosis and are considering manipulating their quantities for therapeutic purposes.

Looking where no one had looked before, Ambros, Baulcombe, and Ruvkun spied an unforeseen universe of potent molecules. Their work has elevated these hitherto unrecognized agents into the spotlight of biology and medicine.

by Evelyn Strauss, Ph.D.

2014 Gruber Genetics Prize
Trio honored for pioneering discoveries of microRNAs
By Jim Fessenden
UMass Medical School Communications

Victor R. Ambros, PhD, professor of molecular medicine, has been awarded the 2014 Gruber Genetics Prize, along with longtime collaborator Gary Ruvkun, PhD, professor of genetics at Massachusetts General Hospital and Harvard Medical School, and David Baulcombe, PhD, professor of botany at the University of Cambridge. They received the prize for their pioneering discoveries of the existence and function of microRNAs and small interfering RNAs, molecules that are now known to play a critical role in gene expression. Dr. Ambros is the Silverman Chair in Natural Sciences and co-director of the RNAi Therapeutics Institute.

Gary Ruvkun, PhD, was awarded the Breakthrough Prize in Life Sciences on November 9, along with Victor Ambros for their work on the discovery of microRNAs and their broad use in biology.

The Breakthrough Prize Foundation announced the recipients of the 2015 Breakthrough Prizes in Fundamental Physics and Life Sciences. These distinguished winners, along with previously announced recipients in the Mathematics category, each receive a $3 million prize.

https://breakthroughprize.org/?controller=Page&action=news&news_id=21

 

Gary Ruvkun, PhD, of the Center for Computational and Integrative Biology and the Department of Molecular Biology, has been awarded the 2014 Gruber Genetics Prize from the Gruber Foundation through Yale University for his work with Victor Ambros, PhD, University of Massachusetts, identifying the existence of microRNAs in animals that control the activity of other genes.

http://gruber.yale.edu/genetics/2014/gary-ruvkun

 

Phillip A. Sharp, PhD
Koch Institute Professor of Integrative Cancer Research

The Sharp Lab focuses on the biology and technology of small RNAs and other types of non-coding RNAs.  RNA interference (RNAi) has dramatically expanded the possibilities for genotype/phenotype analysis in cell biology and for therapeutic intervention.  MicroRNAs (miRNAs) are encoded by endogenous genes and regulate primarily at the stage of translation over half of all genes in mammalian cells.  The Sharp laboratory is working to identify physically the target mRNAs for particular miRNAs.  His laboratory has recently discovered a new class of microRNAs that are produced from sequences adjacent to transcription start sites (TSS-miRNAs).  The functions of the small RNAs are a subject of investigation.  His laboratory is also investigating the relationship between gene regulation by miRNAs and angiogenesis and cellular stress.  Most promoters and enhancers in mammalian cells are transcribed divergently with RNA polymerases initiating in both directions.  Divergent transcription generates thousands of long non-coding RNAs.  The extent of elongation by polymerase in either the sense direction or the antisense direction is controlled by recognition of the nascent RNA by U1 snRNP, a spliceosome component.  The function of the divergent non-coding transcripts is being investigated as well as the relationship of RNA splicing, chromatin modifications and transcription.

 

Noncoding RNAs: A Cache of Cancer Clues?

Rummaging through the Noncoding RNA Attic Has Brought to Light Interesting Baubles—miRNAs and lncRNAs

Kathy Liszewski    GEN Sep  1, 2015 (Vol. 35, No. 15)
http://www.genengnews.com/gen-articles/noncoding-rnas-a-cache-of-cancer-clues/5561/

 

At Weill Cornell Medical College, researchers discovered that estrogen receptors can hijack the androgen-signaling pathway to promote prostate cancer growth. In particular, they found that the estrogen receptor can activate NEAT1, a long noncoding RNA. NEAT1 target genes were determined to be upregulated in several prostate cancer datasets.

http://www.genengnews.com/Media/images/Article/thumb_Cornell_graphics1_Neat1Signature1436235247.jpg

 

At Weill Cornell Medical College, researchers discovered that estrogen receptors can hijack the androgen-signaling pathway to promote prostate cancer growth. In particular, they found that the estrogen receptor can activate NEAT1, a long noncoding RNA. NEAT1 target genes were determined to be upregulated in several prostate cancer datasets.

In the postgenomic era, the numerous and diverse noncoding RNA species once dismissed as “junk RNA” are increasingly seen as treasure. Noncoding RNAs, we now know, have diverse functions in health and disease.

Some in the field believe that we have only started to appreciate the riches of noncoding RNA. The ultimate jewels? They may prove to be previously hidden connections with cancer.

Almost as numerous as newly discovered RNA baubles are the newly organized RNA conferences. One such event, Molecular and Cellular Biology: MicroRNAs and Noncoding RNAs in Cancer, was held June 7–12 in Keystone, CO. This event, a Keystone Symposia conference, focused on the complex universe of RNA biology that is disturbed in cancer.

Providing a perspective on the field was John L. Rinn, Ph.D., an associate professor of stem cell and regenerative biology, Harvard Medical School. He said that if you are not reading a new textbook, your ideas about RNA may be wrong.

“This is a dynamic and fast-moving field,” he insisted. “Recent advances in RNA sequencing technologies have disclosed the existence of thousands of previously unknown noncoding transcripts, including many long noncoding RNAs (lncRNAs) whose functions remain mostly undetermined. However, there are an increasing number of examples that show they are not only key regulators of gene expression, but also direct targets of cancer pathways.”

The laboratory of John L. Rinn, Ph.D., at Harvard Medical School has been studying the role of large intervening ncRNAs (lincRNAs) in establishing the distinct epigenetic states of adult and embryonic cells and their mis-regulation in diseases such as cancer.

http://www.genengnews.com/Media/images/Article/thumb_Harvard_oncolncRNA1512422915.jpg

 

The laboratory of John L. Rinn, Ph.D., at Harvard Medical School has been studying the role of large intervening ncRNAs (lincRNAs) in establishing the distinct epigenetic states of adult and embryonic cells and their mis-regulation in diseases such as cancer.

Noncoding RNAs include the well-known microRNAs (miRNAs) and the lesser-known lncRNAs. Usually defined on the basis of their size, the single-stranded short miRNAs consist of about 22 nucleotides. They regulate gene expression via translation inhibition or degradation of their mRNA targets. Long ncRNAs refer to transcripts that consist of more than 200 nucleotides and lack extended open reading frames. This arbitrary cutoff excludes most known, yet still poorly understood, classes of small RNAs, such as tRNAs and short interfering RNAs.

Recent studies have provided an intriguing hypothesis: Long ncRNAs may be the missing links in cancer. According to Dr. Rinn, “We now know that lncRNAs constitute an important layer of genome regulation over a diverse array of biological processes and diseases, such as cancer.”

Since the ultimate cause of cancer is altered homeostasis of cellular networks and gene expression programs, even the slightest perturbation of these pathways can result in malignant cellular transformation. “These cell circuits are fine-tuned and largely maintained by the coordinated functioning of proteins as well as ncRNAs,” explained Dr. Rinn. “But, beyond the layer of the well-known protein-coding RNAs and miRNAs, lies the realm of lncRNAs that are fast emerging as critical components and regulators of tumor-suppressor and oncogenic pathways.”

Regulator of Metastasis

A precancerous lesion imaged at the University of Minnesota shows abnormal duct morphology and cell proliferation in the mammary gland of a 10-week-old mouse engineered with a single copy number increase of Myc and Pvt1. Gain of Myc alone does not produce such a phenotype.

http://www.genengnews.com/Media/images/Article/thumb_UnivMN_Precancerous1072313061.jpg

A precancerous lesion imaged at the University of Minnesota shows abnormal duct morphology and cell proliferation in the mammary gland of a 10-week-old mouse engineered with a single copy number increase of Myc and Pvt1. Gain of Myc alone does not produce such a phenotype.

The major specific hallmarks of cancer include malignant cell migration, invasion, and metastasis. The latter is the primary cause of cancer recurrence and subsequent death.

“Deregulated lncRNAs may impact a diverse array of human cancers, especially their progression,” said David L. Spector, Ph.D., a professor at the Cold Spring Harbor Laboratory. “One of these lncRNAs is the cancer-associated MALAT1 [metastasis-associated lung adenocarcinoma transcript 1]. It’s not only very abundant in many types of human cells; it is also highly conserved across many mammalian species.”

Dr. Spector’s laboratory identified a novel mechanism for 3′-end processing of this nucleus-restricted lncRNA and is dissecting its mechanism of action: “Since MALAT1 is upregulated in several human cancers, it may play an important role during tumor progression. Because its physiological function at the tissue and organismal levels was unknown, we developed a Malat1 loss-of-function genetic mouse model. Since our in vivo studies demonstrated that Malat1 isn’t essential for mouse development and does not affect global gene expression, we are currently pursuing whether this is due to redundancy or context dependency.”

The team of Sven Diederichs at the German Cancer Research Center DKFZ, in collaboration with the Spector lab, examined the role of MALAT1 by knocking it out in human lung tumor cells. They incorporated an RNA-destabilizing element using zinc finger nucleases. This resulted in a unique loss-of-function model with more than a 1,000-fold silencing. When these cells were utilized in a xenograft mouse model, they found that MALAT1-deficient cells had impaired migration and homing to the lungs. This study supports a role of MALAT1 as a regulator of cell migration that is important in gene expression governing the metastasis of lung cancer cells.

These findings have therapeutic implications, according to Dr. Spector. “MALAT1 could represent a predictive marker of disease and use of antisense oligonucleotides could provide a potential therapeutic strategy,” he concluded.

To extend these studies, Dr. Spector’s group is now examining how altered levels of MALAT1 might impact breast cancer initiation and progression.

Noncoding RNAs: A Cache of Cancer Clues?

Rummaging through the Noncoding RNA Attic Has Brought to Light Interesting Baubles—miRNAs and lncRNAs

http://www.genengnews.com/gen-articles/noncoding-rnas-a-cache-of-cancer-clues/5561/

Cornell_graphics1_Neat1Signature1436235247

http://www.genengnews.com/Media/images/Article/Cornell_graphics1_Neat1Signature1436235247.jpg

At Weill Cornell Medical College, researchers discovered that estrogen receptors can hijack the androgen-signaling pathway to promote prostate cancer growth. In particular, they found that the estrogen receptor can activate NEAT1, a long noncoding RNA. NEAT1 target genes were determined to be upregulated in several prostate cancer datasets.

  • In the postgenomic era, the numerous and diverse noncoding RNA species once dismissed as “junk RNA” are increasingly seen as treasure. Noncoding RNAs, we now know, have diverse functions in health and disease.
  • Some in the field believe that we have only started to appreciate the riches of noncoding RNA. The ultimate jewels? They may prove to be previously hidden connections with cancer.
  • Almost as numerous as newly discovered RNA baubles are the newly organized RNA conferences. One such event, Molecular and Cellular Biology: MicroRNAs and Noncoding RNAs in Cancer, was held June 7–12 in Keystone, CO. This event, a Keystone Symposia conference, focused on the complex universe of RNA biology that is disturbed in cancer.
  • Providing a perspective on the field was John L. Rinn, Ph.D., an associate professor of stem cell and regenerative biology, Harvard Medical School. He said that if you are not reading a new textbook, your ideas about RNA may be wrong.
  • “This is a dynamic and fast-moving field,” he insisted. “Recent advances in RNA sequencing technologies have disclosed the existence of thousands of previously unknown noncoding transcripts, including many long noncoding RNAs (lncRNAs) whose functions remain mostly undetermined. However, there are an increasing number of examples that show they are not only key regulators of gene expression, but also direct targets of cancer pathways.”

The laboratory of John L. Rinn, Ph.D., at Harvard Medical School has been studying the role of large intervening ncRNAs (lincRNAs) in establishing the distinct epigenetic states of adult and embryonic cells and their mis-regulation in diseases such as cancer.

  • Noncoding RNAs include the well-known microRNAs (miRNAs) and the lesser-known lncRNAs. Usually defined on the basis of their size, the single-stranded short miRNAs consist of about 22 nucleotides. They regulate gene expression via translation inhibition or degradation of their mRNA targets. Long ncRNAs refer to transcripts that consist of more than 200 nucleotides and lack extended open reading frames. This arbitrary cutoff excludes most known, yet still poorly understood, classes of small RNAs, such as tRNAs and short interfering RNAs.
  • Recent studies have provided an intriguing hypothesis: Long ncRNAs may be the missing links in cancer. According to Dr. Rinn, “We now know that lncRNAs constitute an important layer of genome regulation over a diverse array of biological processes and diseases, such as cancer.”
  • Since the ultimate cause of cancer is altered homeostasis of cellular networks and gene expression programs, even the slightest perturbation of these pathways can result in malignant cellular transformation. “These cell circuits are fine-tuned and largely maintained by the coordinated functioning of proteins as well as ncRNAs,” explained Dr. Rinn. “But, beyond the layer of the well-known protein-coding RNAs and miRNAs, lies the realm of lncRNAs that are fast emerging as critical components and regulators of tumor-suppressor and oncogenic pathways.”
  • Regulator of Metastasis

A precancerous lesion imaged at the University of Minnesota shows abnormal duct morphology and cell proliferation in the mammary gland of a 10-week-old mouse engineered with a single copy number increase of Myc and Pvt1. Gain of Myc alone does not produce such a phenotype.

A Dangerous PartnershipThe major specific hallmarks of cancer include malignant cell migration, invasion, and metastasis. The latter is the primary cause of cancer recurrence and subsequent death.

  • “Deregulated lncRNAs may impact a diverse array of human cancers, especially their progression,” said David L. Spector, Ph.D., a professor at the Cold Spring Harbor Laboratory. “One of these lncRNAs is the cancer-associated MALAT1 [metastasis-associated lung adenocarcinoma transcript 1]. It’s not only very abundant in many types of human cells; it is also highly conserved across many mammalian species.”
  • Dr. Spector’s laboratory identified a novel mechanism for 3′-end processing of this nucleus-restricted lncRNA and is dissecting its mechanism of action: “Since MALAT1 is upregulated in several human cancers, it may play an important role during tumor progression. Because its physiological function at the tissue and organismal levels was unknown, we developed a Malat1 loss-of-function genetic mouse model. Since our in vivo studies demonstrated that Malat1 isn’t essential for mouse development and does not affect global gene expression, we are currently pursuing whether this is due to redundancy or context dependency.”
  • The team of Sven Diederichs at the German Cancer Research Center DKFZ, in collaboration with the Spector lab, examined the role of MALAT1 by knocking it out in human lung tumor cells. They incorporated an RNA-destabilizing element using zinc finger nucleases. This resulted in a unique loss-of-function model with more than a 1,000-fold silencing. When these cells were utilized in a xenograft mouse model, they found that MALAT1-deficient cells had impaired migration and homing to the lungs. This study supports a role of MALAT1 as a regulator of cell migration that is important in gene expression governing the metastasis of lung cancer cells.
  • These findings have therapeutic implications, according to Dr. Spector. “MALAT1 could represent a predictive marker of disease and use of antisense oligonucleotides could provide a potential therapeutic strategy,” he concluded.
  • To extend these studies, Dr. Spector’s group is now examining how altered levels of MALAT1 might impact breast cancer initiation and progression.

One lncRNA, PVT1, is keeping bad company, at least according to new studies linking it to the key cancer-causing oncogene, MYC. This unexpected partnership has stirred up much interest in the scientific community, especially since MYC is linked to a majority of human cancers.

Anindya Bagchi, Ph.D., an assistant professor of genetics, cell biology and development, University of Minnesota, reported that her group began by looking at structural alterations in cancer genome. “[Of particular interest is the loss or gain of particular segments of the genome that occurs recurrently in cancer,” he notes. “One such region that is of immense interest to us is 8q24, a genomic region often found to be gained in a number of cancers.

“The well-characterized myelocytomatosis (MYC) oncogene resides in the 8q24.21 region. We found that in cancer, MYC is consistently co-gained with an adjacent ‘gene desert’ of about 2 megabases that includes the lncRNA gene PVT1.”

Dr. Bagchi and colleagues utilized chromosomal engineering in mice to construct three iterations to model: MYC only, MYC plus this surrounding area, and the surrounding region alone. “Surprisingly, we found that MYC enhanced tumor growth only when the surrounding region was included,” Dr. Bagchi pointed out. “This verified that MYC is not acting alone.

“We next utilized primary human cancer cell lines and found that PVT1 RNA and MYC protein expression were correlated. Further, we determined that copy number of PVT1 was increased in more than 98% of cancers with MYC gain.”

Finally, Dr. Bagchi’s group definitively fingered PVT1 as the co-conspirator with MYC. The investigators knocked it out of MYC-driven colon cancer cells and found the tumors virtually disappeared. According to Dr. Bagchi, this study complements previous studies and establishes an important finding: Long ncRNA PVT1 interacts with MYC in the nucleus and protects the MYC protein from degradation, probably by reducing phosphorylation of its threonine 58 residue.

“What makes this finding so exciting is that we now may have a much needed tool to target the notoriously elusive MYC protein that has been refractory to small-molecule inhibition,” asserted Dr. Bagchi. “Perhaps by uncoupling this dangerous partnership and targeting PVT1, we could remove the driver that amplifies a major cancer gene.”

  • Prostate Cancer and Noncoding RNA

Given the roles played by ncRNAs in a host of biological processes, it is no surprise that these species also impact prostate cancer progression and therapy resistance. Nonetheless, details of the relationship between ncRNAs and prostate cancer remain to be elucidated, said Dimple Chakravarty, Ph.D., an assistant professor of pathology and laboratory medicine at Weill Cornell Medical College.

“Deregulated or aberrant expression of steroid nuclear receptors are linked with cancer progression and thus are also major targets for therapeutic intervention,” observed Dr. Chakravarty. “But specific therapies are often inadequate.

“For example, the androgen receptor [AR] plays a central role in this malignant progression. Despite the initial effectiveness of therapeutic androgen ablation, resistance inevitably develops to both first generation anti-androgen therapies and to second-generation AR-targeted therapies. The reasons for this are unclear.”

Dr. Chakravarty and colleagues wanted to better understand the role of the estrogen receptor alpha (ERα) that is expressed in prostate cancers. “Our studies identified an ERα-specific noncoding transcriptome signature. This lured us into the noncoding world,” she disclosed.

Dr. Chakravarty and her collaborators, including Mark A Rubin, M.D., a professor of pathology and laboratory medicine at Weill Cornell, scrutinized a combination of chromatin immunoprecipitation (ChIP) and RNA-sequencing data. The investigators found that the most significantly overexpressed and ERα-regulated lncRNA in prostate cancer samples was a transcript called NEAT1, the nuclear enriched abundant transcript 1.

“Our studies utilized a battery of approaches,” detailed Dr. Chakravarty. “We used qRT-PCR and RNA-ISH to examine NEAT1 mRNA levels in prostate cancer tissue and in cell lines, and we analyzed public datasets of normal versus prostate cancer with advanced disease. Epigenetic studies demonstrated that NEAT1 is recruited to the chromatin of prostate cancer genes and contributes to an epigenetic ‘on’ state.”

 

Dr. Chakravarty expressed excitement over these findings: “This study is the first of its kind to demonstrate transcriptional regulation of lncRNAs by an alternative steroid receptor in prostate cancer. We believe NEAT1 could serve as both a prognostic marker for aggressive prostate cancer and also a potential therapeutic target.

 

“Completed and ongoing studies suggest NEAT1 is a good marker for patient risk stratification and a predictor of therapy resistance. We are now exploring the possibility of knocking it out in vivo to see if there is a therapeutic benefit. It could be that targeting NEAT1 and the androgen receptor in combination may provide a unique treatment strategy for a subset of patients who have advanced prostate cancer.”

  • Mouse Models for Noncoding RNA

Genetically engineered mouse models of human cancer have been indispensable in dissecting the molecular mechanisms involved in tumorigenesis. They also provide powerful platforms for preclinically studying drug sensitivity and resistance, said Andrea Ventura, M.D., Ph.D., a cancer biologist at the Memorial Sloan Kettering Cancer Center.

“Mouse models can explore the physiological function of microRNAs such as determining how they affect development and their response to tumor treatments. It is almost impossible to do these studies otherwise,” explained Dr. Ventura. “Another way mouse models are important is for modeling noncoding RNA.”

 

Tools for Studying and Using Small RNAs: From Pathways to Functions to Therapies
This poster provides an overview of the tools that have been developed to understand the functions of small RNAs and, conversely, the use of small RNAs as tools. Tools that are based on small RNAs have been exploited to investigate gene function in cultured cells and in living animals. Small RNA biogenesis, discovery and functional roles are explored in detail.

Read Full Post »


Commentary on Biomarkers for Genetics and Genomics of Cardiovascular Disease: : Views by Larry H Bernstein, MD, FCAP

 

Author: Larry H Bernstein, MD, FCAP

This review has examined a compendium of well regarded documents drawn from 248 articles in Circulation Cardiovascular Genetics from March 2010 to March 2013. The large amount of evidence obtained from large population studies identifying Genome Wide Analysis Studies (GWAS) examines a host of cardiac and vascular diseases in which there is association between specific single nucleotide peptides (SNPs), and gene loci, that may play or have no significant role in developing heart disease. It certainly is evidence of the role that the American Heart Association has is in supporting the leading research today for tomorrow’s patients.   It is too early to sort them out, but it speaks to a large volume of discovery in this area.

It raises another issue that we have been confronted with mostly since the second half of the 20th century.  What is that issue?  The issue, it appears to me, is the vast improvements in analytical technology so that “imprecision” is far less likely to be a confounder in biological measurements and this lends access to far better accuracy?  But from that question arises another! Accuracy only refers to what is measured, but does it give us better ability to explain a complex and dynamic process?  In other words, what is what we are looking at representative of in manageable events?   I think that this is the most important idea that should come out of the recent criticism of the trajectory that molecular genetics been on in the last 5 years.

It was still in an era that “BIG’ science was not the normal.  One could spend an enormous effort at stepwise purification of a protein or enzyme, or other biomolecule starting with a slurry made from 100 lbs of “chicken heart”, for example.  These separations were based on negative charges on the molecules and positive charges on the column, and the molecules of no interest were eluted by gradient elution.  Much was learned about large scale preparation from small scale trials.  But this work was not undertaken without the intent to carry out a number of investigations to understand the “functionality” of a link in a metabolic pathway.  The studies that followed the purification required kinetic investigation with a coenzyme, or with a synthetically modified coenzyme, amino acid sequencing, NMR studies, etc.  You could not put together a “mechanism” without having the minimum amount of necessary information for a reliable account.  It is probably this requirement that led to today’s “BIG” science, that is founded upon multiple methods, now large data bases, and teams of investigators across institutions and continents.  The acquisition of knowledge has been astounding, but the integration of knowledge has not caught up.

However, let’s see if we can sort out the most meaningful signals from what I too am beginning to call the “noisy channel”.  As often happens, important areas of research are opened up that are followed by significant discovery and, in the long run, many other dead end publications that have no lasting significance.  In order to do justice to the work, I’ll pick through documents I find interesting, keeping in mind there is a hidden layer of complexity of which only sufficient information leads to a better understanding.  As much literature calls attention to, much of what ails us has nothing to do with classical Mendelian genetics, and has a postgenomic component.

The most fascinating aspect of this is the withering “dark matter” of the genome. While that component may be silent or expressed, the understanding comes at a higher observed order.  The dark became light! The expression became subtle, like weak bond interactions. The underlying organization is a component of the adaptive ability of an organism or individual in an environment with plants and animals in a changing climate, at particular altitudes, with given water supplies, with disease vectors, and with endogenous sources of essential nutrients.  This brings into focus the regulatory role of the genome as just as important a factor as transmission of the genetic code, especially in somatic cell populations.

The remainder of this discussion deals specifically with my observations on cardiovascular genomics. The following conclusion is appropriate, if incomplete, at this time on circulating miRNAs, particularly miR-133a:

  • elevated levels of circulating miR-133a in patients with cardiovascular diseases originate mainly from the injured myocardium.
  • Circulating miR-133a can be used as a marker for cardiomyocyte death, and
  • it may have functions in cardiovascular diseases.

Circulation: Cardiovascular Genetics. 2011;4:446-454.

Strikingly, in plasma from

  • acute myocardial infarction patients, cardiac myocyte–associated miR-208b and -499 were highly elevated, 1600-fold (P<0.005) and 100-fold (P<0.0005), respectively, as compared with control subjects. Receiver operating characteristic curve analysis revealed an area under the curve of 0.94 (P<10−10) for miR-208b and 0.92 (P<10−9) for miR-499. BothmicroRNAs correlated with plasma troponin T, indicating release of microRNAs from injured cardiomyocytes.
  • In patients with acute heart failure, only miR-499 was significantly elevated (2-fold), whereas
  • no significant changes in microRNAs studied could be observed in diastolic dysfunction.

Remarkably, plasma microRNA levels were not affected by a wide range of clinical confounders, including

  • age,
  • sex,
  • body mass index,
  • kidney function,
  • systolic blood pressure, and
  • white blood cell count.

This is miRNA with a different twist.  It appears that there are 3 types found in AMI(133a, 208b, 409).  But type 409 alone is increased with acute heart failure (no mention of chronic cardiomyopathy and no effect of estimated GFR, or of age).

If the problem was just of AMI, then we have to know what this brings to the table.  As it is the hs-troponins have yet to be shown to effectively not only increase the high sensitivity of the tests, but to decrease the confusion generated by the elevation.  The enormous improvement of a test that may be superior to the hs-ctn’s is for the patient with very indeterminiate shortness of breath, a nondefinitive ECG, and in a prodromal phase of AMI.  This happened in the past, and it may happen now, and it may account for many cases of silent MI that were found at autopsy.

Cited by
Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure Eur J Heart Fail. 2013;0:hft018v1-hft018,


Circulating microRNAs as diagnostic biomarkers for cardiovascular diseases   Am. J. Physiol. Heart Circ. Physiol.. 2012;303:H1085-H1095,

Circulation Editors’ Picks: Most Read Articles in Cardiovascular Genetics Circulation. 2012;126:e163-e169,

MicroRNAs in Patients on Chronic Hemodialysis (MINOS Study) CJASN. 2012;7:619-623,

Novel techniques and targets in cardiovascular microRNA research Cardiovasc Res. 2012;93:545-554,

Microparticles: major transport vehicles for distinct microRNAs in circulationCardiovasc Res. 2012;93:633-644,

Profiling of circulating microRNAs: from single biomarkers to re-wired networksCardiovasc Res. 2012;93:555-562,

Small but smart–microRNAs in the centre of inflammatory processes during cardiovascular diseases, the metabolic syndrome, and ageing   Cardiovasc Res. 2012;93:605-613,

Circulation: Heart Failure Editors’ Picks: Most Important Papers in Pathophysiology and Genetics Circ Heart Fail. 2012;5:e32-e49

Use of Circulating MicroRNAs to Diagnose Acute Myocardial Infarction   Clin. Chem. 2012;58:559-567,

Circulating microRNAs to identify human heart failure   Eur J Heart Fail. 2012;14:118-119,

Next Steps in Cardiovascular Disease Genomic Research–Sequencing, Epigenetics, and Transcriptomics  Clin. Chem. 2012;58:113-126,

Most Read in Cardiovascular Genetics on Biomarkers, Inherited Cardiomyopathies and Arrhythmias, Metabolomics, and GenomicsCirc Cardiovasc Genet. 2011;4:e24-e30,

MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+/Lin- progenitor cells in ischaemia  Cardiovasc Res. 2011;92:449-455,

The use of genomics for treatment is another matter, and has several factors, e.g., age, residual function after AMI, comorbidities

This is a lot of interesting work that opens as many questions as it answers. The observations are real, and they lead to questions relating to the heart and the circulation.  Maybe it will generate answers to very tough issues concerning hypertension, renal disease and the heart.  It is far too early to tell.  It appears that we are about to hear a cacophony of miR’s in a symphony on cardiac and circulatory diseases not be be pieced together soon. But we have many more tools at our disposal than we did when Karmen discovered and made a distinction between

  • Aspartate and Alanine aminotransferases in the late 1950s, followed in the 1960s by
  • Creatine phosphokinase, the
  • MB-isoenzyme of CK by Sobel, Shell and Kjeckshus,
  • isoenzyme-1 of lactate dehydrogenase, and later the
  • Troponins,

leading to the programs to “reduce the extent of infarct damage”.

Then came the

  • and B-type natriuretic peptides (BNP),

which are still not fully understood in their role in congestive heart failure and inrenal disease.

One item strikes the imagination as a fruitful area of further study.   Genetic Determinants of Potassium Sensitivity and Hypertension.    Integrated Computational and Experimental Analysis of the Neuroendocrine Transcriptome in Genetic Hypertension Identifies Novel Control Points for the Cardiometabolic Syndrome

Essential hypertension, a common complex disease, displays substantial genetic influence. Contemporary methods to dissect the genetic basis of complex diseases such as the genomewide association study are powerful, yet a large gap exists betweens the fraction of population trait variance explained by such associations and total disease heritability.

Revised 7/17/2014
 Gene expression profiles associated with acute myocardial infarction and risk of cardiovascular deathJ Kim, NGhasemzadeh, DJEapen, NC Chung, JD Storey,AAQuyyumi and GGibsonKim et al. Genome Medicine 2014, 6:40http://genomemedicine.com/content/6/5/40

Abstract

Background: Genetic risk scores have been developed for coronary artery disease and atherosclerosis, but are not predictive of adverse cardiovascular events. We asked whether peripheral blood expression profiles may be predictive of acute myocardial infarction (AMI) and/or cardiovascular death.

Methods: Peripheral blood samples from 338 subjects aged 62 ± 11 years with coronary artery disease (CAD) were analyzed in two phases (discovery N = 175, and replication N = 163), and followed for a mean 2.4 years for cardiovascular death. Gene expression was measured on Illumina HT-12 microarrays with two different normalization procedures to control technical and biological covariates. Whole genome genotyping was used to support comparative genome-wide association studies of gene expression. Analysis of variance was combined with receiver operating curve and survival analysis to define a transcriptional signature of cardiovascular death.

Results: In both phases, there was significant differential expression between healthy and AMI groups with overall down-regulation of genes involved in T-lymphocyte signaling and up-regulation of inflammatory genes. Expression quantitative trait loci analysis provided evidence for altered local genetic regulation of transcript abundance in AMI samples. On follow-up there were 31 cardiovascular deaths. A principal component (PC1) score capturing covariance of 238 genes that were differentially expressed between deceased and survivors in the discovery phase significantly predicted risk of cardiovascular death in the replication and combined samples (hazard ratio = 8.5, P< 0.0001) and improved the C-statistic (area under the curve 0.82 to 0.91, P= 0.03) after adjustment for traditional covariates.

Conclusions: A specific blood gene expression profile is associated with a significant risk of death in Caucasian subjects with CAD. This comprises a subset of transcripts that are also altered in expression during acute myocardial infarction.

MicroRNA References

Lecture Contents delivered at Koch Institute for Integrative Cancer Research, Summer Symposium 2014: RNA Biology, Cancer and Therapeutic Implications, June 13, 2014 @MIT    Curator of Lecture Contents: Aviva Lev-Ari, PhD, RN https://pharmaceuticalintelligence.wordpress.com/wp-admin/post.php?post=23174&action=edit

3:15 – 3:45, 6/13/2014, Laurie Boyer “Long non-coding RNAs: molecular regulators of cell fate”
https://pharmaceuticalintelligence.com/2014/06/13/315-345-2014-laurie-boyer-long-non-coding-rnas-molecular-regulators-of-cell-fate/

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure. Dickinson BA, Semus HM, Montgomery RL, Stack C, Latimer PA, et al.  Eur J Heart Fail. 2013 Jun; 15(6):650-9.  http://dx.doi.org:/10.1093/eurjhf/hft018

Circulating microRNAs – Biomarkers or mediators of cardiovascular disease?  S Fichtlscherer, AM Zeiher, S Dimmeler. Arteriosclerosis, Thrombosis, and Vascular Biology.2011; 31:2383-2390.
http://dx.doi.org:/10.1161/​ATVBAHA.111.226696

Circulating microRNAs as diagnostic biomarkers for cardiovascular diseases. AJ Tijsen, YM Pinto, and EE Creemers. Am J Physiol Heart Circ Physiol 303: H1085–H1095, 2012.  http://dx.doi.org:/10.1152/ajpheart.00191.2012.

MicroRNAs in Patients on Chronic Hemodialysis (MINOS Study). Emilian C, Goretti E, Prospert F, Pouthier D, Duhoux P, et al. Clin J Am Soc Nephrol  (CJASN)2012;  7: 619-623. http://dx.doi.org:/10.2215/CJN.10471011

Plasma microRNAs serve as biomarkers of therapeutic efficacy and disease progression in hypertension-induced heart failure.BA Dickinson, HM Semus, RL Montgomery, C Stack, PA Latimer, et al.
Eur J Heart Fail 2013 Jun 6;15(6):650-9. http://www.pubfacts.com/detail/23388090/Plasma-microRNAs-serve-as-biomarkers-of-therapeutic-efficacy-and-disease-progression-in-hypertension

Circulating MicroRNAs: Novel Biomarkers and Extracellular Communicators in Cardiovascular Disease?  Esther E. Creemers, Anke J. Tijsen, Yigal M. Pinto.  Circulation Research. 2012; 110: 483-495    http://dx.doi.org:/10.1161/​CIRCRESAHA.111.247452

Novel techniques and targets in cardiovascular microRNA research.  Dangwal S, Bang C, Thum T.Cardiovasc Res. 2012 Mar 15; 93(4):545-54.  http://dx.doi.org:/10.1093/cvr/cvr297

Microparticles: major transport vehicles for distinct microRNAs in circulation. Diehl P, Fricke A, Sander L, Stamm J, Bassler N, Htun N, et al.  Cardiovasc Res. 2012 Mar 15; 93(4):633-44. http://dx.doi.org:/10.1093/cvr/cvs007.

Profiling of circulating microRNAs: from single biomarkers to re-wired networks. A  ZampetakiP Willeit, I Drozdov, S Kiechl and M Mayr. Cardiovasc Res 2012; 93 (4): 555-562.  http://dx.doi.org:/10.1093/cvr/cvr266

Small but smart–microRNAs in the centre of inflammatory processes during cardiovascular diseases, the metabolic syndrome, and ageing. Schroen B, Heymans SCardiovasc Res. 2012; 93(4):605-613.  http://dx.doi.org:/10.1093/cvr/cvr268

Therapeutic Inhibition of miR-208a Improves Cardiac Function and Survival During Heart Failure.  RL Montgomery, TG Hullinger, HM Semus, BA Dickinson, AG Seto, et al.
http://dx.doi.org:/10.1161/​CIRCULATIONAHA.111.030932

Circulating microRNAs to identify human heart failure.  Seto AG, van Rooij E.
Eur J Heart Fail. 2012;14(2):118-119.  http://dx.doi.org:/10.1093/eurjhf/hfr179.

Use of Circulating MicroRNAs to Diagnose Acute Myocardial Infarction.  Y Devaux, M Vausort, E Goretti, PV Nazarov, F Azuaje. Clin Chem. 2012; 58:559-567.  http://dx.doi.org:/10.1373/clinchem.2011.173823

Next Steps in Cardiovascular Disease Genomic Research–Sequencing, Epigenetics, and Transcriptomics  RB Schnabel, A Baccarelli, H Lin, PT Ellinor, and EJ Benjamin.
Clin Chem . 2012 Jan; 58(1): 113–126.  http://dx.doi.org:/10.1373/clinchem.2011.170423

MicroRNA-133 Modulates the {beta}1-Adrenergic Receptor Transduction Cascade.  A Castaldi, T Zaglia, V Di Mauro, P Carullo, G Viggiani, et al.  Circ. Res..2014; 115:273-283.
http://dx.doi.org:/10.1161/​CIRCRESAHA.115.303252

Development of microRNA therapeutics is coming of age.  E van Rooij, S Kauppinen.  EMBOMol Med.. 2014; 6:851-864.  http://dx.doi.org:/10.15252/emmm.201100899

Pitx2-microRNA pathway that delimits sinoatrial node development and inhibits predisposition to atrial fibrillation.   J Wang, Y Bai, N Li, W Ye, M Zhang,et al. PNAS 2014; 111: 9181-9186.
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1405411111/-/DCSupplemental.

MicroRNA-126 modulates endothelial SDF-1 expression and mobilization of Sca-1+/Lin- progenitor cells in ischaemia  Cardiovasc Res. 2011; 92:449-455,
http://dx.doi.org:/10.1093/cvr/cvr227

The use of genomics for treatment is another matter, and has several factors, e.g., age, residual function after AMI, comorbidities

 

 

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