Small but mighty RNAs

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Small but mighty RNAs

September 5, 2015 by larryhbern

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, Gï¿½ttingen) 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

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

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

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

Kathy Liszewski

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

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

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.”

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 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.