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Loss-of-function mutations in a gene called Pax5 have been known to drive normal blood cells to turn into leukemia cells. Such mutations are permanent, so it remained unclear whether an initial, temporary loss of function would instigate an irreversible cascade of events leading to an accumulation of undifferentiated lymphoblasts, or whether an ongoing loss of function would be needed to maintain the disease state.
With the publication of a new study, the question has become more than academic. The study, by researchers at Melbourne’s Walter and Eliza Hall Institute, has not only shown that switching off Pax5 causes cancer in a murine model of B-progenitor acute lymphoblastic leukemia (B-ALL), it has also demonstrated that switching on Pax5 essentially cures the disease.
The results of the study appeared June 15 in the journal Genes & Development, in an article entitled “Pax5 loss imposes a reversible differentiation block in B-progenitor acute lymphoblastic leukemia.” The article described how the researchers used transgenic RNAi to reversibly suppress endogenous Pax5 expression in the hematopoietic compartment of mice, which cooperates with activated signal transducer and activator of transcription 5 (STAT5) to induce B-ALL.
“In this model, restoring endogenous Pax5 expression in established B-ALL triggers immunophenotypic maturation and durable disease remission by engaging a transcriptional program reminiscent of normal B-cell differentiation,” wrote the authors. “Notably, even brief Pax5 restoration in B-ALL cells causes rapid cell cycle exit and disables their leukemia-initiating capacity.”
Institute researcher Grace Liu noted that Pax5, which is frequently “lost” in childhood B-ALL, is essential for normal development of B cells. “When Pax5 function is compromised, developing B cells can get trapped in an immature state and become cancerous,” she said. “We have shown that restoring Pax5 function, even in cells that have already become cancerous, removes this ‘block,’ and enables the cells to develop into normal white blood cells.”
Simply restoring Pax5 sufficed to normalize cancer cells. That is, re-engaging the stalled differentiation program in immature white blood cells restored normal development “despite the presence of additional oncogenic lesions.”
Institute researcher Ross Dickins, Ph.D., said that forcing B-ALL cells to resume their normal development could provide a new strategy for treating leukemia: “While B-ALL has a relatively good prognosis compared with other cancers, current treatments can last years and have major side effects. By understanding how specific genetic changes drive B-ALL, it may be possible to develop more specific treatments that act faster with fewer side effects.”
“It is very difficult to develop drugs that restore the function of genes that are lost during cancer development,” Dr. Dickins added. “However, by understanding the mechanisms by which Pax5 loss causes leukemia, we can begin to look at ways of developing drugs that could have the same effect as restoring Pax5 function.”
Pax5 is just one of about 100 genes known to suppress human tumors. Now that Pax5 has been scrutinized with genetic switch technology, the researchers speculate that similar technology could be used to characterize other tumor suppressor genes.
Loss of Gene Islands May Promote a Cancer Cell’s Survival, Proliferation and Evolution: A new Hypothesis (and second paper validating model) on Oncogenesis from the Elledge Laboratory
Writer, Curator: Stephen J. Williams, Ph.D.
It is well established that a critical event in the transformation of a cell to the malignant state involves the mutation of hosts of oncogenes and tumor suppressor genes, which in turn, confer on a cell the inability to properly control its proliferation. On a genomic scale, these mutations can result in gene amplifications, loss of heterozygosity (LOH), and epigenetic changes resulting in tumorigenesis. The “two hit hypothesis”, proposed by Dr. Al Knudson of Fox Chase Cancer Center[1], proposes that two mutations in the same gene are required for tumorigenesis, initially proposed to explain the progression of retinoblastoma in children, indicating a recessive disease.
(Excerpts from a great article explaining the two-hit-hypothesis is given at the end of this post).
And, although many tumor genomes display haploinsufficeint tumor suppressor genes, and fit the two hit model quite nicely, recent data show that most tumors display hemizygous recurrent deletions within their genomes. Tumors display numerous recurrent hemizygous focal deletions that seem to contain no known tumor suppressor genes. For instance a recent analysis of over three thousand tumors including breast, bladder, pancreatic, ovarian and gastric cancers averaged greater than 10 deletions/tumor and 82 regions of recurrent focal deletions,
It has been proposed these great number of hemizygous deletions may be a result of:
a recessive tumor suppressor gene requiring mutation or silencing of second allele
the mutation may recur as they are located in fragile sites (unstable genomic regions)
single-copy loss may provide selective advantage regardless of the other allele
Note: some definitions of hemizygosity are given below. In general at any locus, each parental chromosome can have 3 deletion states:
wild type
large deletion
small deletion
Hemizygous deletions only involve one allele, not both alleles which is unlike the classic tumor suppressor like TP53
To see if it is possible that only one mutated allele of a tumor suppressor gene may be a casual event for tumorigenesis, Dr. Nicole Solimini and colleagues, from Dr. Stephen Elledge’s lab at Harvard, proposed a hypothesis they termed the cancer gene island model, after analyzing the regions of these hemizygous deletions for cancer related genes[2]. Dr. Soliin and colleagues analyzed whole-genome sequence data for 526 tumors in the COSMIC database comparing to a list generated from the Cancer Gene Census for homozygous loss-of-function mutations (mutations which result in a termination codon or frame-shift mutation: {this produces a premature stop in the protein or an altered sequence leading to a nonfunctional protein}.
Results of this analysis revealed:
although tumors have a wide range of deletions per tumor (most epithelial high grade like ovarian, bladder, pancreatic, and esophageal adenocarcinomas had 10-14 deletions per tumor
and although tumors exhibited a wide range (2- 16 ) loss of function mutations
ONLY 14 of 82 recurrent deletions contained a known tumor suppressor gene and was a low frequency event
Most recurrent cancer deletions do not contain putative tumor suppressor genes.
Therefore, as the authors suggest, an alternate method to the two-hit hypothesis may account for a selective growth advantage for these types of deletions, defining these low frequency hemizygous mutations in two general classes
STOP genes: suppressors of tumor growth and proliferation
GO genes: growth enhancers and oncogenes
Identifying potential STOP genes
To identify the STOP and GO genes the authors performed a primary screen of an shRNA library in telomerase (hTERT) immortalized human mammary epithelial cells using increased PROLIFERATION as a screening endpoint to determine STOP genes and decreased proliferation and lethality (essential genes) to determine possible GO genes. An initial screen identified 3582 possible STOP genes. Using further screens and higher stringency criteria which focused on:
Only genes which increased proliferation in independent triplicate screens
Validated by competition assays
Were enriched more than four fold in three independent shRNA screens
the authors were able to focus on and validate 878 genes to determine the molecular pathways involved in proliferation.
These genes were involved in cell cycle regulation, apoptosis, and autophagy (which will be discussed in further posts).
To further validate that these putative STOP genes are relevant in human cancer, the list of validated STOP genes found in the screen was compared to the list of loss-of-function mutations in the 526 tumors in the COSMIC database. Surprisingly, the validated STOP gene list were significantly enriched for known and possibly NOVEL tumor suppressor genes and especially loss of function and deletion mutations but also clustered in gene deletions in cancer. This not only validated the authors’ model system and method but suggests that hemizygous deletions in multiple STOP genes may contribute to tumorigenesis
as the function of the majority of STOP genes is to restrain tumorigenesis
A few key conclusions from this study offer strength to an alternative view of oncogenesis NAMELY:
Loss of multiple STOP genes per deletion optimize a cancer cell’s proliferative capacity
Cancer cells display an insignificant loss of GO genes, minimizing negative impacts on cellular fitness
Haploinsufficiency in multiple STOP genes can result in similar alteration of function similar to complete loss of both alleles of
Cancer evolution may result from selection of hemizygous loss of high number of STOP and low number of GO genes
Leads to a CANCER GENE ISLAND model where there is a clonal evolution of transformed cells due to selective pressures
A link to the supplemental data containing STOP and GO genes found in validation screens and KEGG analysis can be found at the following link:
A concern of the authors was the extent to which gene silencing could have on their model in tumors. The validation of the model was performed in cancer cell lines and compared to tumor genome sequence in publicly available databases however a followup paper by the same group shows that haploinsufficiency contributes a greater impact on the cancer genome than these studies have suggested.
In a follow-up paper by the Elledge group in the journal Cell[3], Theresa Davoli and colleagues, after analyzing 8,200 tumor-normal pairs, show there are many more cancer driver genes than once had been predicted. In addition, the distribution and potency of STOP genes, oncogenes, and essential genes (GO) contribute to the complex picture of aneuploidy seen in many sporadic tumors. The authors proposed that, together with these and their previous findings, that haploinsufficiency plays a crucial role in shaping the cancer genome.
Zygosity is the degree of similarity of the alleles for a trait in an organism.
Most eukaryotes have two matching sets of chromosomes; that is, they are diploid. Diploid organisms have the same loci on each of their two sets of homologous chromosomes, except that the sequences at these loci may differ between the two chromosomes in a matching pair and that a few chromosomes may be mismatched as part of a chromosomal sex-determination system. If both alleles of a diploid organism are the same, the organism is homozygous at that locus. If they are different, the organism is heterozygous at that locus. If one allele is missing, it is hemizygous, and, if both alleles are missing, it is nullizygous.
Haploinsufficiency occurs when a diploid organism has only a single functional copy of a gene (with the other copy inactivated by mutation) and the single functional copy does not produce enough of a gene product (typically a protein) to bring about a wild-type condition, leading to an abnormal or diseased state. It is responsible for some but not all autosomal dominant disorders.
Al Knudsen and The “Two-Hit Hypothesis” of Cancer
Excerpt from a Scientist article by Eugene Russo about Dr. Knudson’s Two hit Hypothesis;
The “two-hit” hypothesis was, according to many, among the more significant milestones in that rapid evolution of biomedical science. The theory explains the relationship between the hereditary and nonhereditary, or sporadic, forms of retinoblastoma, a rare cancer affecting one in 20,000 children. Years prior to the age of gene cloning, Knudson’s 1971 paper proposed that individuals will develop cancer of the retina if they either inherit one mutated retinoblastoma (Rb) gene and incur a second mutation (possibly environmentally induced) after conception, or if they incur two mutations or hits after conception.3 If only one Rb gene functions normally, the cancer is suppressed. Knudson dubbed these preventive genes anti-oncogenes; other scientists renamed them tumor suppressors.
When first introduced, the “two-hit” hypothesis garnered more interest from geneticists than from cancer researchers. Cancer researchers thought “even if it’s right, it may not have much significance for the world of cancer,” Knudson recalls. “But I had been taught from the early days that very often we learn fundamental things from unusual cases.” Knudson’s initial motivation for the model: a desire to understand the relationship between nonhereditary forms of cancer and the much rarer hereditary forms. He also hoped to elucidate the mechanism by which common cancers, such as those of the breast, stomach, and colon, become more prevalent with age.
According to the then-accepted somatic mutation theory, the more mutations, the greater the risk of cancer. But this didn’t jibe with Knudson’s own studies on childhood cancers, which suggested that, in the case of cancers such as retinoblastoma, disease onset peaks in early childhood. Knudson set out to determine the smallest number of cancer-inducing events necessary to cause cancer and the role of these events in hereditary vs. nonhereditary cancers. Based on existing data on cancer cases and some mathematical deduction, Knudson came up with the “two-hit” hypothesis.
Not until 1986, when researchers at the Whitehead Institute for Biomedical Research in Cambridge, Mass., cloned the Rb gene, would there be solid evidence to back up Knudson’s pathogenesis paradigm.4 “Even with the cloning of the gene, it wasn’t clear how general it would be,” says Knudson. There are, it turns out, several two-hit lesions, including polyposis, neurofibromitosis, and basal cell carcinoma syndrome. Other cancers show only some correspondence with the two-hit model. In the case of Wilm’s tumor, for example, the model accounts for about 15 percent of the cancer incidence; the remaining cases seem to be more complicated.
A.G. Knudson, “Mutation and cancer: statistical study of retinoblastoma,” Proceedings of the National Academy of Sciences, 68:820-3, 1971.
The two hit hypothesis proposed by A.G. Knudson. A description with video of Dr. Knudson talk at AACR can be found at the following link (photo creditied to A.G. Knudson and Fox Chase Cancer Center at the following link:http://www.fccc.edu/research/research-awards/knudson/index.html
2. Solimini NL, Xu Q, Mermel CH, Liang AC, Schlabach MR, Luo J, Burrows AE, Anselmo AN, Bredemeyer AL, Li MZ et al: Recurrent hemizygous deletions in cancers may optimize proliferative potential. Science 2012, 337(6090):104-109.
3. Davoli T, Xu Andrew W, Mengwasser Kristen E, Sack Laura M, Yoon John C, Park Peter J, Elledge Stephen J: Cumulative Haploinsufficiency and Triplosensitivity Drive Aneuploidy Patterns and Shape the Cancer Genome. Cell 2013, 155(4):948-962.
Other papers on this site on CANCER and MUTATION include:
Researchers have been unable to explain why cancer cells contain abnormal numbers of chromosomes for over a century. The phenomenon known as aneuploidy is associated with all types of cancer. Harvard Medical School researchers have hypothesized why cancer cells contain many more chromosome abnormalities than healthy cells. They have devised a way to understand
patterns of aneuploidy in tumors and
predict which genes in the affected chromosomes are likely to be cancer suppressors or promoters, and
they propose that aneuploidy is a driver of cancer, rather than a result of it.
The study, to be published online in Cell, offers a new theory of cancer development and could lead to new treatment targets. This would be feasible if they could identify key cancers suppressors.
The cancer cell characteristically has many gene deletions and amplifications, chromosome gains and losses. Although it has the appearance of randomness, previous research has shown that there is a pattern to the alterations in chromosomes and chromosome arms, which suggests that we can decipher that pattern and perhaps learn how or if it drives the cancer, according to the senior author, Stephen Elledge, Gregor Mendel professor of Genetics and of Medicine at HMS and professor of medicine at Brigham and Women’s Hospital. Having proposed the theory about how these cellular genetic changes occur, the team set out to prove it using mathematical analysis.
See “Related Links” for full-size image. (Source: HMS/ University of Cambridge/Joanne Davidson, Mira Grigorova and Paul Edwards)
Mining for answers
Cancer research has focused on mutations for decades since the “oncogene revolution.” Changes in the DNA code that abnormally activate genes, called oncogenes, either promote cancer or deactivate genes that suppress cancer. The role of aneuploidy— in which entire chromosomes or chromosome arms are added or deleted— has remained largely unstudied.
Elledge and his team, including research fellow and first author Teresa Davoli, suspected that aneuploidy has a significant role to play in cancer because missing or extra chromosomes likely affect genes involved in tumor-related processes such as cell division and DNA repair.
To test their hypothesis, the researchers developed a computer program called TUSON (Tumor Suppressor and Oncogene) Explorer together with Wei Xu and Peter Park at HMS and Brigham and Women’s. The program analyzed genome sequence data from more than 8,200 pairs of cancerous and normal tissue samples in three preexisting databases.
They found many more potential cancer drivers than anticipated
after generating a list of suspected oncogenes and tumor suppressor genes based on their mutation patterns.
They ranked the suspects by how powerful an effect their deletion or duplication was likely to have on cancer development. The team then looked at where the suspects normally appear in chromosomes.
They discovered that
the number of tumor suppressor genes or oncogenes in a chromosome
correlated with how often the whole chromosome or part of the chromosome was deleted or duplicated in cancers.
Where there were concentrations of tumor suppressor genes alongside
fewer oncogenes and fewer genes essential to survival,
there was more chromosome deletion.
Conversely,
concentrations of oncogenes and fewer tumor suppressors coincided with
When the team factored in gene potency, the correlations got even stronger. A cluster of highly potenttumor suppressors was
more likely to mean chromosome deletion than a cluster of weak suppressors.
Number matters
Since 1971, the standard tumor suppressor model has held that
cancer is caused by a “two-hit” cascade in which first one copy and
then the second copy of a gene becomes mutated.
Elledge argues that simply losing or gaining one copy of a gene through aneuploidy can influence tumor growth as well. However, the loss or gain of multiple cancer driver genes that individually have low potency
can have big effects by accretion of potency
These novel algorithms that identify tumor suppressors and oncogenes give experimentally verifiable basis for how aneuploidies evolve in cancer cells, and
Indicate that subtle changes in the activity of many different genes at the same time can contribute to tumorigenesis
These findings also may have answered a long-standing question about whether aneuploidy is a cause or effect of cancer, leaving researchers free to pursue the question of how. “Aneuploidy is driving cancer, not simply a consequence of it,” said Elledge. “Other things also matter, such as gene mutations, rearrangements and changes in expression. We don’t know what the weighting is, but now we should be able to figure it out.” Elledge and Davoli plan to gather experimental evidence to support their mathematical findings. That will include validating some of the new predicted tumor suppressors and oncogenes as well as “making some deletions and amplifications and seeing if they have the properties we think they do”.
Long Noncoding RNA Network regulates PTEN Transcription
Author: Larry H Bernstein, MD, FCAP
Scientists Find Surprising New Influence On Cancer Genes
A pseudogenelong noncoding RNA networkregulates PTEN transcription and translation in human cells
Per Johnsson, A Ackley, L Vidarsdottir, Weng-Onn Lui, M Corcoran, D Grandér, and KV Morris
Mol Cancer. 2011; 10: 38. Published online 2011 April 13. doi: 10.1186/1476-4598-10-38 PMCID: PMC3098824
New Type of Gene That Regulates Tumour Suppressor PTEN Identified
Feb. 24, 2013 — Researchers at Karolinska Institutet in Sweden have identified a new so-called pseudogene that regulates the tumour-suppressing PTEN gene.
They hope that this pseudogene will be able to control PTEN to
reverse the tumour process,
make the cancer tumour more sensitive to chemotherapy and
The development of tumours coincides with the activation of several cancer genes as well as the inactivation of other tumour-suppressing genes owing to
damage to the DNA and
to the fact that
the cancer cells manage to switch offthe transcription of tumour-suppressor genes.
To identify what might be regulating this silencing, the researchers studied PTEN,
one of the most commonly inactivated tumour-suppressor genes.
It has long been believed that the switching-off process is irreversible, but the team has now shown that
silenced PTEN genes in tumour cells can be ‘rescued’ and
re-activated by a ‘pseudogene’,
a type of gene that, unlike normal genes,
does not encode an entire protein.
“We identified a new non-protein encoding pseudogene, which
determines whether the expression of PTEN
is to be switched on or off,”
says research team member Per Johnsson, at Karolinska Institutet’s Department of Oncology-Pathology. “What makes this case spectacular is that the gene
only produces RNA,
the protein’s template.
It is this RNA that, through a sequence of mechanisms,
regulates PTEN.
Pseudogenes have been known about for many years, but
it was thought that they were only junk material.”
No less than 98 per cent of human DNA consists of non-protein encoding genes (i.e. pseudogenes), and by studying these formerly neglected genes the researchers
have begun to understand that they are very important and
can have an effect without encoding proteins.
Using model systems, the team has shown that the new pseudogene can
control the expression of PTEN and
make tumours more responsive to conventional chemotherapy.
Pre Johnssom suggests “we might one day be able to re-programme cancer cells
to proliferate less,
become more normal, and that
resistance to chemotherapy can hopefully be avoided.
“We also believe that our findings can be very important for the future development of cancer drugs. The human genome conceals no less than 15,000 or so pseudogenes, and it’s not unreasonable to think
that many of them are relevant to diseases such as cancer.”
The study was conducted in collaboration with scientists at The Scripps Research Institute, USA, and the University of New South Wales, Australia, and was made possible with
grants from the Swedish Childhood Cancer Foundation, the Swedish Cancer Society, the Cancer Research Funds of Radiumhemmet, Karolinska Institutet’s KID programme for doctoral studies, the Swedish Research Council, the Erik and Edith Fernström Foundation for Medical Research, the National Institute of Allergy and Infectious Diseases, the National Cancer Institute and the National Institutes of Health.
The functional role of long non-coding RNA in human carcinomas EA Gibb, CJ Brown, and WL Lam Long non-coding RNAs (lncRNAs) are emerging as new players in the cancer paradigm demonstrating potential roles in both oncogenic and tumor suppressive pathways. These novel genes are frequently
aberrantly expressed in a variety of human cancers,
however the biological functions of the vast majority remain unknown. Recently, evidence has begun to accumulate describing the molecular mechanisms by which these RNA species function, providing insight into
the functional roles they may play in tumorigenesis.
In this review, we highlight the emerging functional role of lncRNAs in human cancer.
One of modern biology’s great surprises was the discovery that the human genome encodes only ~20,000 protein-coding genes, representing <2% of the total genome sequence [1,2]. However, with the advent of
tiling resolution genomic microarrays and
whole genome and transcriptome sequencing technologies
it was determined that at least 90% of the genome is actively transcribed [3,4].
The human transcriptome was found to be more complex than
a collection of protein-coding genes and their splice variants; showing
extensive antisense,
overlapping and non-coding RNA (ncRNA) expression [5-10].
Although initially argued to be spurious transcriptional noise, recent evidence suggests that the proverbial “dark matter” of the genome
may play a major biological role in cellular development and metabolism [11-17].
One such player, the newly discovered long non-coding RNA (lncRNA) genes, demonstrate
developmental and tissue specific expression patterns, and
aberrant regulation in a variety of diseases, including cancer [18-27].
NcRNAs are loosely grouped into two major classes based on transcript size; small ncRNAs and lncRNAs [28-30].
Small ncRNAs are represented by a broad range of known and newly discovered RNA species, with many being associated
with 5′ or 3′ regions of genes [4,31,32].
This class includes the well-documented miRNAs, RNAs ~22 nucleotides (nt) long involved in the specific regulation of both
protein-coding, and
putatively non-coding genes,
by post-transcriptional silencing or infrequently
by activation [33-35].
miRNAs serve as major
regulators of gene expression and as
intricate components of the cellular gene expression network [33-38].
Another newly described subclass are the transcription initiation RNAs (tiRNAs), which are
the smallest functional RNAs at only 18 nt in length [39,40].
small ncRNAs classes, including miRNAs, have established roles in tumorigenesis, an intriguing association between
the aberrant expression of ncRNA satellite repeats and cancer has been recently demonstrated [41-46].
Types of human non-coding RNAs
In contrast to miRNAs, lncRNAs, the focus of this article, are
mRNA-like transcripts ranging in length from 200 nt to ~100 kilobases (kb) lacking significant open reading frames.
Many identified lncRNAs are transcribed by RNA polymerase II (RNA pol II) and are polyadenylated, but this is not a fast rule [47,48].
There are examples of lncRNAs, such as the
antisense asOct4-pg5 or the
brain-associated BC200,
which are functional, but not polyadenylated [49-51].
lncRNA expression levels appear to be lower than protein-coding genes [52-55], and some
lncRNAs are preferentially expressed in specific tissues [21].
Novel lncRNAs may contribute a significant portion of the aforementioned ‘dark matter’ of the human transcriptome [56,57]. In an exciting report
by Kapranov et.al., it was revealed the bulk of the relative mass of RNA in a human cell, exclusive of the ribosomal and mitochondrial RNA,
is represented by non-coding transcripts with no known function [57].
Like miRNAs and protein-coding genes, some
transcriptionally active lncRNA genes display
histone H3K4 trimethylation at their 5′-end and
histone H3K36 trimethylation in the body of the gene [8,58,59].
The small number of characterized human lncRNAs have been associated with a spectrum of biological processes, for example,
epigenetics,
alternative splicing,
nuclear import,
as structural components,
as precursors to small RNAs and
even as regulators of mRNA decay [4,60-70].
Furthermore, accumulating reports of misregulated lncRNA expression across numerous cancer types suggest that
aberrant lncRNA expression may be a major contributor to tumorigenesis [71].
This surge in publications reflects the increasing attention to this subject and a number of useful lncRNA databases have been created .
In this review we highlight the emerging
functional role of aberrant lncRNA expression, including
transcribed ultraconserved regions (T-UCRs), within human carcinomas.
Publications describing cancer-associated ncRNAs. Entries are based on a National Library of Medicine Pubmed search using the terms
“ncRNA” or “non-coding RNA” or “noncoding RNA” or non-protein-coding RNA” with cancer and annual (Jan.1-Dec.31) date limitations. …
Publically available long non-coding RNA online databases
The definition ‘non-coding RNA’ is typically used to describe transcripts where
sequence analysis has failed to identify an open reading frame.
There are cases where ‘non-coding’ transcripts were found to encode short, functional peptides [72]. Currently, a
universal classification scheme to define lncRNAs does not exist. Terms such as
large non-coding RNA,
mRNA-like long RNA, and
intergenic RNA
all define cellular RNAs, exclusive of rRNAs,
greater than 200 nt in length and having no obvious protein-coding capacity [62].
This has led to confusion in the literature as to exactly which transcripts should constitute a lncRNA. One subclass of lncRNAs is called
large or long intergenic ncRNAs (lincRNAs). These lncRNAs are
exclusively intergenic and are
marked by a chromatin signature indicative of transcription [8,58].
RNA species that are bifunctional preclude categorization into either group of
protein-coding or
ncRNAs as
their transcripts function both at the RNA and protein levels [73].
The term ‘lncRNA‘ is used only to describe transcripts with no protein-coding capacity. In the meantime, and for the purposes of this review,
we will consider lncRNAs as a blanket term to encompass
mRNA-like ncRNAs,
lincRNAs, as well as
antisense and intron-encoded transcripts,
T-UCRs and
transcribed pseudogenes.
Discovery of LncRNAs
The earliest reports describing lncRNA predated the discovery of miRNAs, although the term ‘lncRNA‘ had not been coined at the time .
One of the first lncRNA genes reported was the imprinted H19 gene, which was quickly followed by the discovery of the
silencing X-inactive-specific transcript (XIST) lncRNA gene, which
plays a critical function in X-chromosome inactivation [74,75].
The discovery of the first miRNA lin-14 dramatically redirected the focus ofncRNA research from long ncRNAs to miRNAs [76], and
the discovery of miRNAs revealed RNA could
regulate gene expression and
entire gene networks could be affected by ncRNA expression and
Within the last decade miRNAs were discovered to be associated with cancer. At the time of this writing there are approximately
1049 human miRNAs described in miRBase V16 [80,81] with the potential of
affecting the expression of approximately 60% of protein -coding genes [82,83].
Conversely, the variety and dynamics of lncRNA expression was not to be fully appreciated until the introduction of whole transcriptome sequencing.
With the advent of the FANTOM and ENCODE transcript mapping projects, it was revealed that the mammalian genome is extensively transcribed,
although a large portion of this represented non-coding sequences [3,84]. Coupled with the novel functional annotation of a few lncRNAs, this discovery
promoted research focusing on lncRNAdiscovery and characterization. Recent reports have described new lncRNA classes such as lincRNAs and T-UCRs [8,58,85].
Current estimates of the lncRNA gene content in the human genome ranges from ~7000 – 23,000 unique lncRNAs, implying this class of ncRNA will
represent an enormous, yet undiscovered, component of normal cellular networks that may be disrupted in cancer biology [62].
Emerging Role of Long Non-Coding RNA in Tumorigenesis
A role for differential lncRNA expression in cancer had been suspected for many years, however, lacked strong supporting evidence [86]. With advancements
in cancer transcriptome profiling and accumulating evidence supporting lncRNA function, a number of differentially expressed lncRNAs have been associated
with cancer. LncRNAs have been implicated to
regulate a range of biological functions and
the disruption of some of these functions, such as
genomic imprinting and transcriptional regulation,
plays a critical role in cancer development.
Here we describe some of the better characterized lncRNAs that have been associated with cancer biology.
Human cancer-associated lncRNAs
Imprinted lncRNA genes
Imprinting is a process whereby the copy of a gene inherited from one parent is epigenetically silenced [87,88]. Intriguingly, imprinted regions often
include multiple maternal and paternally expressed genes with a high frequency of ncRNA genes. The imprinted ncRNA genes are implicated in the
imprinting of the region by a variety of mechanisms including
enhancer competition and chromatin remodeling [89].
A key feature of cancer is the loss of this imprinting resulting in altered gene expression [90,91]. Two of the best known imprinted genes
are in fact lncRNAs.
H19
The H19 gene encodes a 2.3 kb lncRNA that is expressed exclusively from the maternal allele. H19 and its reciprocally imprinted protein-coding neighbor
the Insulin-Like Growth Factor 2 or IGF2 gene at 11p15.5 were among the first genes, non-coding or otherwise, found to demonstrate genomic imprinting [74,92].
The expression of H19 is high during vertebrate embryo development, but is
downregulated in most tissues shortly after birth with the exception of skeletal tissue and cartilage [20,93,94].
Loss of imprinting and subsequent strong gene expression has been well-documented in human cancers. Likewise,
loss of imprinting at the H19 locus resulted in high H19 expression in cancers of the esophagus, colon, liver, bladder and with hepatic metastases [95-97].
H19 has been implicated as having both oncogenic and tumor suppression properties in cancer. H19 is upregulated in a number of human cancers, including
hepatocellular, bladder and breast carcinomas, suggesting an oncogenic function for this lncRNA [97-99]. In colon cancer H19 was shown to be directly activated
by the oncogenic transcription factorc-Myc, suggesting
H19 may be an intermediate functionary between c-Myc and downstream gene expression [98].
Conversely, the tumor suppressor gene and transcriptional activatorp53 has been shown to
down-regulate H19 expression [100,101].
H19 transcripts also serve as a precursor for miR-675, a miRNA involved in the regulation of developmental genes [102]. miR-675 is processed from the first exon of H19 and functionally
downregulates the tumor suppressor gene retinoblastoma (RB1) in human colorectal cancer, further implying an oncogenic role for H19 [103].
There is evidence suggesting H19 may also play a role in tumor suppression [104,105]. Using a mouse model for colorectal cancer, it was shown that mice lacking H19 manifested an increased polyp count compared to wild-type [106]. Secondly, a mouse teratocarcinoma model demonstrated larger
tumor growth when the embryo lacked H19, and finally in a hepatocarcinoma model, mice developed cancer much earlier when H19 was absent [107].
The discrepancy as to whether H19 has oncogenic or tumor suppressive potential may be due in part to the bifunctional nature of the lncRNA or may
be context dependent. In either case, the precise functional and biological role of H19 remains to be determined.
XIST – X-inactive-specific transcript
The 17 kb lncRNA XIST is arguably an archetype for the study of functionallncRNAs in mammalian cells, having been studied for nearly two decades.
In female cells, the XIST transcript plays a critical role in X-chromosomeinactivation by
physically coating one of the two X-chromosomes, and is necessary for the
cis-inactivation of the over one thousand X-linked genes [75,108-110].
Like the lncRNAs HOTAIR and ANRIL, XIST associates with polycomb-repressor proteins, suggesting
a common pathway of inducing silencing utilized by diverse lncRNAs.
A study led by Manel Esteller, director of the Epigenetics and Cancer Biology Program at the Bellvitge Biomedical Research Institute (IDIBELL), professor of genetics at the University of Barcelona and ICREA researcher has completed the first epigenome in Europe.
The finding is published in the journalEpigenetics.
The genome of all cells in the human body is the same for all of them, regardless their aspect and functions. Therefore, genome cannot fully explain the activity of tissues and organs and their disorders in complex diseases like cancer. It takes a further explanation. Part of this explanation is provided by epigenetics, a field of biology that studies the heredity activity of DNA that does not involve changes in its sequence. That is, if genetics is the alphabet, epigenetics is the spelling that guides the activity of our cells.
Methylation
Epigenetics refers to chemical changes in our genetic material and proteins that regulate it. The best-known epigenetic mark is the methylation, the addition of a methyl chemical group (-CH3) in our DNA. The epigenome consists of all the epigenetic marks of a living being. The authors of the study have completed the epigenomes for all brands of methylation of DNA from white blood cells of two girls: a healthy one and a patient suffering from a rare genetic disease called Immunodeficiency, Centromere instability and Facial anomalies syndrome (ICF). This disease is caused by a mutation in a gene that causes the addition of a methyl chemical group in its DNA.
The analysis performed by the researchers reveals that the patient has an epigenomic defect that causes fragility of chromosomes, which thus can easily be broken. In addition, the study shows that the patient has a wrong epigenetic control of many genes related to the response against infection, which causes a severe immune deficiency. The study coordinator, Manel Esteller, emphasizes that due to this study, “we now know what happens in this type of rare diseases and we can start thinking about strategies for new treatments based on this knowledge.”
Dr. Esteller’s work has been crucial to show that all human tumours have in common a specific chemical alteration: the hypermethylation of tumour suppressor genes.
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