Friday, December 14, 2012

PCA3, EZH2, the Androgen Receptor and Control of Survival


Multiple epigenetic markers have been determined as determinants for prognostic values in prostate cancer, PCa. There are two recent papers, one of PCA3 and its pathway control, and on EZH2 and its use as a marker. We briefly summarize these efforts and attempt to place them in a common and ever growing context of both prognostic markers as well as putative pathway control therapeutic targets.

PCA3

PCA3 has received a great deal of attention of late. It is a non-coding RNA and the controlling gene is located at 9q21-q22[1]. It is also called prostate cancer antigen 3 (non-protein coding). The presence of PCA3 is generally now believed to be a marker for PCa. Testing is now underway on may patients to determine if they have PCa using the PCA3 assay. Thus there is a great deal of interest in better understanding what the full networks are for PCA3 generation as well as looking at those pathways as a possible means to control PCa. We examine two recent studies in this area.

In the recent paper by Ferreira et al, they state:

Our findings suggest that the ncRNA PCA3 is involved in the control of PCa cell survival, in
part through modulating AR signaling, which may raise new possibilities of using PCA3
knockdown as an additional therapeutic strategy for PCa control.

This may be of significant merit as a new potentially useful therapeutic. Now it should be recalled that the AR pathway and the PSA generation is known as shown below[2].

Now Ferreira et al continue:

Due to the increased PCA3 expression in androgen-responsive cells compared with androgen-insensitive cells, and because AR signaling is an important pathway controlling PCa survival, we tested whether PCA3 expression was modulated by the androgen-active metabolite DHT and whether this expression pattern involved the activated AR.

Upregulation of PCA3 expression in response to LNCaP stimulation with DHT was significantly counteracted by the AR antagonist flutamide, indicating that PCA3 expression was induced by the activated AR. AR activation was further confirmed by the observation that LNCaP cells stimulated with DHT also showed AR transcriptional activity. Consistently, all of the AR target genes tested that contain canonical AR response elements (AREs) in their promoter sequences, were upregulated upon DHT treatment. Although eight of the genes showed at least a 1.5-fold increase after AR activation, only two of them showed a significant increase in their expression levels. Interestingly, PCA3 upregulation upon DHT treatment has been observed previously, but no study has demonstrated the involvement of activated AR in PCA3 expression by using AR antagonists. Although our data also suggest that PCA3 is an androgen-responsive gene, the precise molecular mechanism by which PCA3 expression responds to this activation is still unknown.

One hypothesis is that activated AR can directly activate the PCA3 promoter, as has been demonstrated for the miR-101 and miR- 21 regulatory regions, which are also modulated by the activated AR. However, no consensus AREs have been identified in the 500-bp PCA3 promoter region. We further screened for consensus ARE elements in the entire PCA3 genomic region at the 5 Kb region upstream from the PCA3 transcription start site, and have so far identified no canonical element (data not shown). Nevertheless, we cannot exclude the possibility that other, noncanonical ARE elements could also promote AR binding and directly activate PCA3 expression, as has been previously described for other genes modulated by the AR activation. PCA3-upregulated expression in response to DHT treatment could also be a result of activated AR binding to the regulatory regions of other AR-responsive genes, which in turn could induce PCA3 expression. Further experiments should investigate direct AR binding to different PCA3 genomic regions, in order to answer these open questions.

Now they examined genes which are known pathway controllers of PCa. The CDKs especially control cell cycle flow.

As an approach to investigate the signal by which PCA3 controls PCa cell survival, we analyzed the transcript expression of PSA, AR, TMPRSS2, NDRG1, GREB1, FGF8, CDK1, CDK2, and PMEPA1 genes, all of which have key roles in PCa growth and progression, and are classical AR target genes.

Also highly regulated by androgens, fibroblast growth factor 8 (FGF8), cyclin-dependent kinase 1 (CDK1), cyclin-dependent kinase 2 (CDK2), and the gene regulated in breast cancer 1 (GREB1) gene products have classical stimulating roles in prostate growth and proliferation. Conversely, the PMEPA1 gene, although a direct transcriptional target of the AR, has been described as a negative regulator of cell growth in the prostate epithelium, as well as negatively regulating AR protein levels in different cell-culture models. We also observed that the AR transcription level was downregulated after PCA3 knockdown. These results accord with previously published data, which demonstrated that the AR gene is transcriptionally regulated by AR through binding to AR regulatory elements (autoregulation). However, differently from the other AR-responsive genes tested here, the ARE elements required for this process have not been found in the AR promoter or in the 5'-flanking region, but rather in AR coding sequences.

The observation that PCA3 is involved in the control by modulation of the AR target genes is a key observation. As we have shown, based upon various prior works, the change in AR is critical to the loss of any control over the PCa cells. They state:

Here we demonstrate for the first time that PCA3 is involved in the control of PCa cell survival, at least in part by modulating the transcriptional activity of AR target genes. To our knowledge, this is the first characterization of the functional role of PCA3 in PCa cells, and will not only improve the understanding of key roles of this transcript in prostate carcinogenesis, but also suggests an alternative strategy to use PCA3 as a putative specific target for PCa treatment approaches. Because PCA3 seems to be a regulator of the expression of AR target genes and PCa cell survival, treatment options aiming to downregulate PCA3, in combination with other androgen-depletion-based strategies, could potentially circumvent androgen-ablation resistance mechanisms.

In an earlier paper by Ferreira et al, they state:

The prostate cancer antigen 3 (DD3/PCA3) is a non-coding RNA (ncRNA) specifically expressed in prostate tissues and overexpressed in prostate cancer (PCa) tumors. Although widely applied as a diagnostic marker for PCa, to date nothing has described about its role in PCa biology. We used herein small interfering RNA (siRNA) in order to knockdown DD3 mRNA message as an approach to elucidate DD3 functional roles in PCa cells.

LNCaP cell line was been used herein as an in vitro model for DD3 functional assays. siRNA sequences were specifically designed for DD3 exon 4 mRNA sequences (siDD3), as well an scrambled siRNA (siScr), as negative control. LNCaP cells were transiently transfected with siDD3 or siScr and DD3 expression was analysed by real time PCR (qRT-PCR) using DD3 specific oligonucleotides. LNCaP cells transfected with siDD3 demonstrated a marked decrease in cell proliferation and viability, as compared to siScr transfected cells.

Further, LNCaP cells in which DD3 was knocked-down presented a significant increase in proportion of cells in SubG0/G1 phase of cell cycle and presenting pyknotic nuclei, indicative of cells undergoing apoptosis. In order to investigate the putative mechanisms underlying the decrease of LNCaP cell survival as a result of DD3 knockdown, we then evaluated the involvement of DD3 on androgen receptor (AR) pro-survival signaling. DD3 expression was significantly uregulated as a result of LNCaP treatment with dihydrotestosterone (DHT), the active androgen metabolite. This effect was reverted by the addition of the AR antagonist, flutamide.

Consistent to an AR activation by DHT treatment, LNCaP cells presented a significant upregulation of AR target genes. Notably, siDD3/LNCaP transfected cells significantly inhibited the expression of tested AR responsive genes. Besides, DD3 knockdown was able to counteract DHT stimulatory effects over AR target gene expression. Despite negatively modulating the transcription of AR target genes, DD3 knockdown did not alter Akt and ERK phosphorylation, suggesting that DD3 is mainly controlling the expression of signaling pathways downstream to AR activation.

In summary, our findings indicate that DD3 is a ncRNA whose expression is AR regulated and is involved on the control of PCa cell survival and proliferation, in part by modulating the AR signaling pathway and its target genes.

These findings correspond to the first description of DD3 roles on PCa cells and could provide new insights into understanding prostate carcinogenesis, besides opening new prospects to use DD3 not only as a biomarker for PCa, but also as an specific target for therapeutic approaches aiming to inhibit PCa growth by negatively modulating AR pro-survival signal and their target genes.

In this slightly earlier paper the authors focus on the PCA3 as a target and examine its pathway significance.

Other researchers have examined PCA3 as well as other markers. It is well known that the TMPRSS2:ERG fusion is often seen in PCA. As Salagierski and Schalken conclude:

In recent years advances in genetics and biotechnology have stimulated the development of noninvasive tests to detect prostate cancer. Serum and urine molecular biomarkers have been identified, of which PCA3 has already been introduced clinically.

The identification of prostate cancer specific genomic aberrations, ie TMPRSS2:ERG gene fusion, might improve diagnosis and affect prostate cancer treatment. Although several recently developed markers are promising, often showing increased specificity for prostate cancer detection compared to that of prostate specific antigen, their clinical application is limited. The only 2 true prostate cancer specific biomarkers identified to date remain PCA3 and TMPRSS2:ERG gene fusion.

Let us briefly summarize these two genes and their fusion.

TMPRSS2:ERG

The TMPRSS2-ERG fusion is the single most seen molecular lesion in prostate cancer. (see Taylor et al 2010) TMPRSS2 is on 21q22.3 and ERG is on 21q22.3. Both are dominant. Unlike the pathway disturbances, this is a fusion, translocation on the same gene, and the resultant is  expressive of ERG and not of TMPRSS2.

Transcriptional regulator ERG is a protein that in humans is encoded by the ERG gene (Ets Related Gene, Chromosome 21).  ERG is a member of the ETS family of transcription factors.
Transcriptional regulator ERG is a nuclear protein that binds purine-rich sequences.  ERG can fuse with TMPRSS2 protein to form an oncogenic fusion gene that is commonly found in human prostate cancer, especially in hormone-refractory prostate cancer.  This suggests that ERG overexpression may contribute to development of androgen-independence in prostate cancer through disruption of androgen receptor signaling.

Transmembrane protease, serine 2 is an enzyme that in humans is encoded by the TMPRSS2 gene. This gene encodes a protein that belongs to the serine protease family. The encoded protein contains a type II transmembrane domain, a receptor class A domain, a scavenger receptor cysteine-rich domain and a protease domain. Serine proteases are known to be involved in many physiological and pathological processes.  This gene was demonstrated to be up-regulated by androgenic hormones in prostate cancer cells and down-regulated in androgen-independent prostate cancer tissue. The protease domain of this protein is thought to be cleaved and secreted into cell media after autocleavage. The biological function of this gene is unknown. TMPRSS2 protein's function in prostate carcinogenesis relies on overexpression of ETS transcription factors, such as ERG and ETV1 through gene fusion. TMPRSS2-ERG fusion gene is the most frequent, present in 40% - 80% of prostate cancers in humans.  

As Weinberg notes:

In the case of the TMPRSS-ERG fusion, both genes are located on 21q22, and the fusion frequently occurs because of an interstitial deletion . The resultant fusion transcripts are androgen responsive and usually encode an ETS gene (ERG) truncated at its N terminus without any coding elements from TMPRSS2. It is unknown if the biologic consequences of misexpression of the truncated ETS family protein are different from expression of the full length protein and whether truncation contributes to oncogenicity. (Ref Weinberg)

There has been a significant amout of research as to the overexpression of PCA3 and why specifically this may be the case. As Auprich et al stat

The PCA3 gene is highly overexpressed in specific PCa cell lines and prostatic tumours. In 2006, a simple and robust urine test (Progensa) became commercially available. Despite its costs, prostate cancer antigen 3 (PCA3) is superior to prostate-specific antigen (PSA) and percent free PSA in the early detection of PCa. PCA3 improves the diagnostic accuracy of externally validated nomograms among men with an elevated PSA undergoing biopsy.

PCA3 independently predicts low-volume disease and pathologically insignificant PCa but is not associated with locally advanced disease and is limited in the prediction of aggressive cancer. Preliminary data demonstrate that combining PCA3 with other new biomarkers further improves diagnostic and prognostic accuracy.

Finally, findings of the first PCA3-Gene-ViroTherapy study suggest therapeutic potential by exploiting PCA3 overexpression. PCA3, integrated in novel biopsy nomograms or risk stratification tools, can be used to counsel or confirm biopsy indications. If confirmed in further studies, using PCA3 together with established staging risk factors could assist clinicians in specific pretreatment decision making. So far no evidence for the usefulness of PCA3 in active surveillance programs has been presented.

The above seems to indicate that although PCA3 is indicative of PCa in low volume states but they state that it is not a metric for high volume states. Other work appears to provide added light of PCA3 and may change this observation.

We look at a recent thesis presented by Lee specifically on a more detailed analysis of PCA3. From Lee we have:

Proposed mechanism of action of PC-TSGC toward the downregulation of signal transduction of Rho GTPase family members. PC-TSGC inhibits the binding of RhoA to its activator Lbc-RhoGEF by direct interaction with RhoA through the BCH domain, and recruits nm23- H1 which in turn inhibits Tiam1, a specific Rac activator. GEF: Guanine nucleotide Exchange Factor; GAP: GTPase Activating Protein.


From Lee p 89 we have:

Proposed biological roles of PCA3 and PC-TSGC in prostate cancer.

(A) Normal cell: growth stimuli are signaled to the nucleus through multiple pathways that include activation of RhoA and Rac and subsequent phosphorylation of AKT and ERK1/2. The signal transduction cascade stimulates gene expression in order to initiate cellular replication and inhibit apoptosis. Simultaneously, the same signals elicit the expression of PC-TSGC which in turn inhibits RhoA and Rac (through nm23-H1), thereby resulting in a negative-feedback loop on the activity of cell growth signaling pathways.

(B) Cancer cell: in a malignant cell, the same mechanism is altered by the abnormal expression of PCA3, which opposes the expression of PC-TSGC. As a result, the control over the RhoA and Rac signaling pathways is lost, and the cell engages an unregulated cell growth that potentially leads to oncogenic transformation. 
Now for abnormal cell growth we have:


EZH2

We now examine another recent marker which also acts in an epigenetic manner, specifically EZH2. EZH2 (located at 7q35-q36) is a member of the Polycomb group, members of which are often associated with the silencing of genes. The epigenetic capabilities allow it to block the expression of multiple genes which are useful in normal cell homeostasis.

As NCBI states[3]:

This gene encodes a member of the Polycomb-group (PcG) family. PcG family members form multimeric protein complexes, which are involved in maintaining the transcriptional repressive state of genes over successive cell generations. This protein associates with the embryonic ectoderm development protein, the VAV1 oncoprotein, and the X-linked nuclear protein. This protein may play a role in the hematopoietic and central nervous systems. Multiple alternatively splcied transcript variants encoding distinct isoforms have been identified for this gene.

PCa can move from Androgen responsive to Androgen resistant by the blocking of certain genetic controls and also the activation of others. Simply we see the three step process as follows:

First the normal cell operation is as shown below:

Then when the cell becomes cancerous, we see the expression of the cancerous genes but they are supported by activated AR products. Often in this stage we still have a localized stage and by depriving the androgen the AR are suppressed in their activation.

Finally we can get to the androgen resistant state as we show below. Several things happen here. First, androgen is actually produced to self-sustain the malignant cell. Second, mutant AR cells can activate independent of the presence of androgens. Third AR proteins can become enhanced with specific sensitivity.  The cell then becomes resistant to any reduction of cell exogenous androgen availability. This stage of PCa then becomes the most aggressive.

As is reported in Science, EZH2 has been seen to have special significance in AR resistant PCa. They state:

Epigenetic regulators are implicated in cancer progression and proposed as therapeutic targets. Xu et al. report that EZH2 (Enhancer of zeste homolog 2), a factor previously thought to exert its oncogenic function primarily as part of the polycomb repressive complex, acts through a distinct mechanism in cells of castration-resistant prostate cancer. Rather than exclusively silencing gene expression through histone methylation, EZH2 acts as a transcriptional coactivator. The activation function of EZH2 plays a critical role in the growth of castration-resistant prostate cancer cells, which could be relevant in future drug development.

Xu and the authors state:

Epigenetic regulators represent a promising new class of therapeutic targets for cancer. Enhancer of zeste homolog 2 (EZH2), a subunit of Polycomb repressive complex 2 (PRC2), silences gene expression via its histone methyltransferase activity. We found that the oncogenic function of EZH2 in cells of castration-resistant prostate cancer is independent of its role as a transcriptional repressor. Instead, it involves the ability of EZH2 to act as a coactivator for critical transcription factors including the androgen receptor. This functional switch is dependent on phosphorylation of EZH2 and requires an intact methyltransferase domain. Hence, targeting the non-PRC2 function of EZH2 may have therapeutic efficacy for treating metastatic, hormone-refractory prostate cancer.

Again a targeting of the AR resistant form of PCa has a potential target in this protein. They conclude:

This study demonstrates that phosphorylation of EZH2 at Ser21, mediated directly or indirectly by the PI3K-Akt pathway, can switch its function from a Polycomb repressor to a transcriptional coactivator of AR (and potentially other factors). Rescue experiments and the lack of correlation with H3K27me3 levels support a role for EZH2-directed methylation of substrates other than H3K27, including potential nonhistone proteins. The current rationale for EZH2 inhibitor design is based primarily on targeting its Polycomb-repressive activity and uses H3K27me3 as the pharmacodynamic readout.

However, the observed loss-of-function mutations of EZH2 inmyelodysplastic syndrome and acute leukemia raise concerns that such inhibitors might exhibit important hematologic side effects.

Our finding of an altered function for EZH2 in CRPC cells raises the potential to develop inhibitors that specifically target the EZH2 activation function while sparing its PRC2-repressive function. In addition, our finding that EZH2 cooperates with AR-associated complexes and requires phosphorylation to support CRPC growth suggests novel combination therapies for the treatment of metastatic, hormonerefractory prostate cancer.

Thus they contend that developing a therapeutic for this specific product could address the AR instabilities.
 
References
 1.               Auprich M, et al, Contemporary role of prostate cancer antigen 3 in the management of prostate cancer, Eur Urol. 2011 Nov;60(5):1045-54. doi: 10.1016/j.eururo.2011.08.003. Epub 2011 Aug 25.
2.               Ferreira, L. et al, DD3/PCA3 non-coding RNA regulates prostate cancer cell survival and modulates AR signaling, Cancer Research: April 15, 2012; Volume 72, Issue 8, Supplement 1 , Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 201., http://cancerres.aacrjournals.org/cgi/content/short/72/8_MeetingAbstracts/201?rss=1 .
3.               Ferreira, L. et al, PCA3 noncoding RNA is involved in the control of prostate-cancer cell survival and modulates androgen receptor signaling, BMC Cancer 2012, 12:507; http://www.biomedcentral.com/content/pdf/1471-2407-12-507.pdf
4.               Lee, A., A NEW TUMOR SUPPRESSOR GENE CANDIDATE REGULATED BY THE NONCODING RNA PCA3 IN HUMAN PROSTATE CANCER, (2010). Univ Texas GSBS Dissertations and Theses, PhD, (Open Access). http://digitalcommons.library.tmc.edu/cgi/viewcontent.cgi?article=1047&context=utgsbs_dissertations
5.               Salagierski M, Schalken JA., Molecular diagnosis of prostate cancer: PCA3 and TMPRSS2:ERG gene fusion. , J Urol. 2012 Mar;187(3):795-801. doi: 10.1016/j.juro.2011.10.133. Epub 2012 Jan 15.
6.               Weinberg, R., Cancer, Garland (New York) 2008.
7.               Xu, K.  et al., EZH2 Oncogenic Activity in Castration-Resistant Prostate Cancer Cells Is Polycomb-Independent, Science 338, 1465 (2012).
 

[2] Note we use the reference, Prostate Cancer Genomics, McGarty (2012, DRAFT, http://www.telmarc.com/Documents/Books/Prostate%20Cancer%20Systems%20Approach%2003.pdf ) as the source for some of this information. From this source one may obtain the initial sources.