Thursday, August 15, 2013

long non-coding RNA (lncRNA) and Prostate Cancer


A recent paper on the understanding of several long non-coding RNAs in the case of androgen resistant prostate cancer has raised the hopes of many to begin understanding the function of these epigenetic players in the control of malignant cells. To date, very few lncRNAs have been determined no less understood functionally. This brief note focuses on them in the context of PCa. We examine the AR case of PCa, then the basics of lncRNAs, and then spend some time examining the recent work and its implications. Finally we present some overall observations.

Androgen Pathway

Prostate cancer can be controlled if not cured if the cancer is detected and removed in a controlled area. However when the cancer begins to spread it lives off of androgens for a long while, and by eliminating the androgens we can in turn “starve” the cancer cells. However the cells manage to find alternative paths to existing without the androgens and this becomes what is termed androgen resistant (AR) prostate cancer, PCa, or ARPCa.

Normal operations of the prostate cell are shown in the Figure below. They result in normal cell homeostasis, namely basal and luminal cells reproducing as needed and in a normal manner.

The next step is a cell becoming cancerous. This we depict below. The result is excess cell growth and loss of apoptosis. Yet the driver is still the androgens entering the cell and driving the process through the AR.

Finally we have the AR independent growth as shown below. The assumption is that mutations occur that result in the ability to activate the AR functions leading to uncontrolled cell growth without the androgen exogenously being provided. In the recent paper in Nature that we shall discuss the growth change and control is now linked to epigenetic elements, namely the lncRNAs.

Long non-coding RNA

Long non-coding RNA, lncRNA, are the long RNAs recently discovered, most of which whose function is yet unknown, which can actually control gene transcription. The lncRNAs range from 200 to well over 100,000 nucleotides. In Weinberg’s latest edition of Cancer he presents about one page only to lncRNAs, and such is an example of their newness and lack of understanding[1].

Kovalchuks state that lncRNA have several functions:

1.     Regulation of expression of neighboring genes
2.     Blocking of splicing proteins-coding genes using antisense transcripts
3.     Interaction with proteins making them more or less capable of fulfilling specific functions
4.     Serving as precursors for smaller ncRNAs.

Kornienko et al present an excellent overview of these functions and we summarize here in their words some key elements of them:

Regulation of transcription is considered to be interplay of tissue and developmental-specific transcription factors (TFs) and chromatin modifying factors acting on enhancer and promoter sequences to facilitate the assembly of the transcription machinery at gene promoters. With a growing number of lncRNAs implicated in transcriptional gene regulation, this view may need refinement to include networks of tissue and developmental-stage specific lncRNAs that complement known regulators to tightly control gene expression and thereby organism complexity.

Transcriptional regulation by lncRNAs could work either in cis or in trans, and could negatively or positively control pc gene expression. lncRNAs work in cis when their effects are restricted to the chromosome from which they are transcribed, and work in trans when they affect genes on other chromosomes.

They continue:

lncRNAs can inhibit general protein-coding (pc) gene expression in trans

(a) by preventing transcription factor (TF) activity (7SK lncRNA) or

(b) by inhibiting RNAPII binding to DNA (B2 lncRNA). Xist lncRNA is transcribed from the X inactivation center (XIC) and inactivates a whole chromosome in cis

(c) by recruiting epigenetic modifiers (EM). lncRNAs can regulate specific genes, acting in trans like HOTAIR

(d) or in cis like HOTTIP

(e) by directly recruiting epigenetic modifiers to certain genomic loci.

In both cases the lncRNA binds EMs via a specific sequence or structure and targets them to promoter regions via DNA/RNA interaction elements to affect expression of the respective pc gene. Transcription of a lncRNA through a pc gene promoter or a cis-regulatory element (RE) affects pc gene expression in cis independent of the lncRNA product (f) by mechanisms discussed in the text. Both DNA strands are shown as separate boxes to indicate lncRNA transcription over the pc gene promoter in the antisense orientation.  

Thus the lncRNAs have become an interesting target for examination especially as we learn more about why certain cancers return after targeted pathway control. lncRNAs are one of the many epigenetic elements which make understanding the process of cancer development and metastasis so complex.
PCa and lncRNA

There is a recent paper in Nature describing how lncRNA acts in an interesting manner of providing ongoing growth capability in the case of androgen resistant prostate cancer, ARPCa. From the overview in Eureka we have:

…the study shows that two long non-coding RNAs (PRNCR1 and PCGEM1) activate androgen receptors, circumventing androgen-deprivation therapy. In their active state, these receptors turn on genes that spur growth and metastasis, making these cancers highly treatment-resistant. The study illustrates how prostate cancer can thrive, even when deprived of hormones, and provides tempting targets for new therapies.

"Androgen-deprivation therapy will often put cancer in remission, but tumors come back, even without testosterone," said contributor Christopher Evans, professor and chair of the Department of Urology at the UC Davis School of Medicine. "We found that these long non-coding RNAs were activating the androgen receptor. When we knocked them out, cancer growth decreased in both cell lines and tumors in animals."

…These prostate cancers are very aggressive and usually fatal, but their continued growth, despite being deprived of hormones, is just now being better understood. It's not unlike removing the key from a car ignition, only to have the vehicle re-start on its own. In this case, the aberrant starting mechanisms are long non-coding RNAs, a class of genetic material that regulates gene expression but does not code for proteins. Using patient samples from UC Davis, the group determined that both PRNCR1 and PCGEM1 are highly expressed in aggressive tumors…

Further investigation determined that one of these long non-coding RNAs is turning on androgen receptors by an alternate switching mechanism, like a car with a second ignition. This is critically important because many prostate cancer treatments work by blocking a part of the androgen receptor called the C-terminus. However, PCGEM1 activates another part of the receptor, called the N-terminus, which also turns on genes — with bad results. "The androgen receptor is unique, if you knock out the C-terminus, that remaining part still has the ability to transcribe genes," said Evans.

In addition, about 25 percent of these cancers have a mutated version of the androgen receptor that has no C-terminus. These receptors are locked in the "on" position, activating genes associated with tumor aggression.

Regardless of the receptor's status, PRNCR1 and PCGEM1 are crucial to prostate cancer growth. In turn, knocking out these RNAs has a profound impact on gene expression, both in cell lines and animal models. The team used complementary genetic material, called antisense, to knock out the RNAs and observe how the tumors and cells responded. In each case, there was a direct relationship between RNA activity, gene expression and cancer growth. "These long non-coding RNAs are a required component for these castration-resistant cancers to keep growing," said Evans. "Now we have preclinical proof of principle that if we knock them out, we decrease cancer growth."

Now we consider the work directly. Ling et al report:

Although recent studies have indicated roles of long non-coding RNAs (lncRNAs) in physiological aspects of cell-type determination and tissue homeostasis1, their potential involvement in regulated gene transcription programs remains rather poorly understood. The androgen receptor regulates a large repertoire of genes central to the identity and behavior of prostate cancer cells2, and functions in a ligand-independent fashion in many prostate cancers when they become hormone refractory after initial androgen deprivation therapy3. Here we report that two lncRNAs highly overexpressed in aggressive prostate cancer, PRNCR1 (also known as PCAT8) and PCGEM1, bind successively to the androgen receptor and strongly enhance both ligand-dependent and ligand-independent androgen-receptor mediated gene activation programs and proliferation in prostate cancer cells.

They continue:

In addition to their relevance to disease, the current results illuminate several fundamental molecular mechanisms. PRNCR1 and PCGEM1 underscore a new role of RNA interaction with sequence-specific DNA-binding proteins — modification of transcription factor activity. The liaisons between lncRNAs and transcription factors can program stepwise chemical modifications on transcription factors, gating the successive flow of information from enhancer engagement to target-gene activation. The insights that these findings provide into how lncRNAs can mediate enhancer–promoter looping are also intriguing. The RNA-mediated recruitment of a protein with intrinsic avidity for a promoter-associated histone mark to distantly located enhancer elements could stabilize DNA looping and promote communication over three-dimensional space. This would mean that, rather than being simple scaffolds, lncRNAs are more akin to a complex computer circuit board, linking together various disparate molecular components and dictating the logical operation of the system.

In a Nature commentary on the paper the authors Schmitt and Change state:

Yang et al. report that two long non-coding RNAs (lncRNAs) — PRNCR1 and PCGEM1 — activate the androgen receptor. Interaction of PRNCR1 with this receptor at androgen-response genomic elements allows recruitment of DOT1L, an enzyme that methylates and so activates the receptor. PCGEM1 can now bind to the active androgen receptor and recruit the enzyme Pygo2, which allows communication between this receptor and its target genes by binding to H3K4me3 chromatin marks in the genes’ promoter sequences. Many androgen receptor target genes have been implicated in prostate-cancer growth.

This discovery is quite useful and insightful. It represents a powerful argument for more aggressively examining the epigenetic factors of the lncRNA. We have seen the impact of miRNA and of methylation and now this opens another powerful area.

This is one of the first papers to lay out a complete story for how the lncRNAs may control metastatic growth. This also is a key element in our growing body of knowledge of epigenetic factors and cancer.

There are several observations worth noting:

1. Classic pathway analysis totally neglects epigenetic factors. This miRNA, lncRNA, and methylation have almost been considered as noise. Must we now expand the model to directly and expressly include these factors and if so how.

2. There is a reference to dealing with the lncRNAs via a therapeutic. There are two questions here; first, what therapeutic and second as we learn from BRAF inhibitors on melanoma and in hypomethylation therapeutics in MDS there are unintended consequences. What may they be as we expand to lncRNA?

3. As we have just begun to touch the edge of the complex and as yet indeterminate number of lncRNAs, how can we deal with them holistically?

4. As one may suspect these may add to our ever growing markers for cancer diagnosis and prognosis. How will these be added?

5. Exosomes are one way to determine what is in a cell. They have been used for prostate cancer staging. Can we now target these lncRNAs in such an exosome test?

1.     Griffith, D., Targeting aggressive prostate cancer, Eureka, , 14 August 2013.
2.     Kornienko, A., et al, Gene regulation by the act of long non-coding RNA transcription, BMC Biology 2013, 11:59.
3.     Kovalchuk, I., O., Kovalchuk, Epigenetics, FT Press (Upper Saddle River, NJ) 2012.
4.     McGarty, T. P., Prostate Cancer Genomics, DRAFT, 2013,
5.     Schmitt, A, H., Chang, Long RNAs wire up cancer growth, Nature, 14 August, 2013.
6.     Shi X, Sun M, Liu H, Yao Y, Song Y. (2013) Long non-coding RNAs: A new frontier in the study of human diseases. Cancer Letters, August 2013.
7.     Sun M, Kraus WL. (2013) Minireview: Long Non-Coding RNAs: New “Links” Between Gene Expression and Cellular Outcomes in Endocrinology. Mol Endocrinol, August 2013.
8.     Weinberg, R., The Biology of Cancer, Garland (New York), 2013.
9.     Yang, L., et al, lncRNA-dependent mechanisms of androgen receptor-regulated gene activation programs, Nature, 14 August, 2013.

[1] Weinberg, 2013, p 26.