One of the most critical tools for assessing prostate cancer in its earlier stages is the potential for the tumor to be or become an aggressive tumor. Most PCa tumors are indolent, growing at a slow rate and often not being the ultimate cause of death. However there are a few PCa which are quite aggressive going from a low level to death in a short period, just two to four years. Being able to identify these tumors is becoming a significant area of study. Morbidity and costs can be significantly reduced if one can identify what cell hold the potential for such aggressive growth.
In a recent paper by Irshad et al presents three markers
that they contend are significant for ascertaining aggressiveness. The genes
and markers are:
1. CDKN1A, a gene which encodes a protein p21. This is a
cyclin dependent kinase (“CDK”) inhibitor. The CDKs function as cell cycle
controls and by inhibiting them the cell cycle, namely proliferation, can be inhibited.
p21 expression is controlled by p53 and by the PI3K/AKT pathway which itself is
controlled by PTEN. Thus failure of either PTEN or p53 can lead to down
regulation of p21 and thus up regulation of cell cycle proliferation. This is a
logical gene product to measure.
2. FGFR1: This is a fibroblast growth factor gene. It has a
powerful impact on angiogenesis and thus can be a significant factor in the
development of blood flow to tumors. Excess expression of the gene may be a
significant factor in malignant angiogenesis. It also is a driver for the EGFR
gene which in turn is a growth factor modulator as well.
3. PMP22: This gene encodes a protein which relates to
peripheral myelin control. Mutations in this gene result in Charcot–Marie–Tooth
disease (CMT), a hereditary neuropathy of the distal joints and muscles. However
PMP22 controls SIVA1 which is a critical gene controlling apoptosis. Thus any
down regulation of this gene would logically down regulate apoptosis and allow
the cell to survive. Combined with the CDKN1A dysregulation we then have a
potentially lethal spiral.
Thus we have three separate and distinct factors which we
summarize below in the graphic.
Thus there may be some significant impact on the
aggressiveness of tumors with aberrant expression of these genes.
We therefore have a putatively deadly spiral which results
in the aggressive form of PCa as we graphically demonstrate below:
The authors summarize their work as follows:
Many newly diagnosed prostate cancers present as low
Gleason score tumors that require no treatment intervention. Distinguishing the
many indolent tumors from the minority of lethal ones remains a major clinical
challenge. We now show that low Gleason score prostate tumors can be
distinguished as indolent and aggressive subgroups on the basis of their
expression of genes associated with aging and senescence. Using gene set
enrichment analysis; we identified a 19-gene signature enriched in indolent
prostate tumors. We then further classified this signature with a decision
tree learning model to identify three genes—FGFR1, PMP22, and CDKN1A—that
together accurately predicted outcome of low Gleason score tumors. Validation
of this three-gene panel on independent cohorts confirmed its independent
prognostic value as well as its ability to improve prognosis with currently
used clinical nomograms. Furthermore, protein expression of this three-gene panel
in biopsy samples distinguished Gleason 6 patients who failed surveillance over
a 10-year period. We propose that this signature may be incorporated into
prognostic assays for monitoring patients on active surveillance to facilitate
appropriate courses of treatment.
One must ask if these genes are the cause of the effect; if
the cause then why and if the effect then what is driving them? We examine some
details herein and discuss the results.
We briefly examine the three genes and their pathways. These
three genes are quite disparate and do not appear to have any common
functionality or proximate causality in cell degeneration.
1. PMP22 is a myelin controlling gene which is connected to Charcot
Marie Tooth disease a disorder of the distal muscles where in there is a
degenerative process resulting in such characteristics as club foot.
2. FGFR is a fibrogen growth receptor which when activated can
create fibrogen.
3. CDKN1A is a cyclin kinase and a significant factor in cell cycle
activation.
Peripheral Myelin Protein 22 (PMP22) is a product of a gene
related to myelin production. In addition a disruption in its function is often
seen in Charcot Marie Tooth disease, a myelo-disruptive disease of the distal
muscles.
As D'Urso et al had stated:
Recent molecular and genetic studies have provided some
insights into the structure and function of one of the integral membrane
proteins of peripheral myelin, the peripheral myelin protein 22 (PMP22). The
pattern of expression of PMP22 is synchronous with myelin formation, and it
localizes almost exclusively in the compact sheath
They conclude by stating:
In summary, our data provide the first direct evidence
for the formation of P0–PMP22 complexes at the plasma membrane. These protein
interactions probably participate in holding adjacent Schwann cell membranes
together and in stabilizing myelin compaction. Our results could explain why
genetic alterations in one of the two partner molecules lead to very similar
disease phenotypes. Normally, a critical number of functional P0 and PMP22 molecules
are necessary to maintain membrane adhesion and myelin compaction. Mutations
could affect the amount of functional PMP22 or P0 in the myelin membrane
through either impaired membrane targeting of the mutated protein or the
disability of the altered protein to establish correct interactions with the
partner molecule because of changes in their conformation. We believe that the
outcome of the present study provides new insight into the molecular basis of
myelin assembly and peripheral dysmyelinating diseases.
As Sereda and Nave state:
The most frequent genetic subtype of Charcot-Marie-Tooth
disease is CMT1A, linked to chromosome 17p11.2. In the majority of cases, CMT1A
is a gene dosage disease associated with a 1.5 Mb large genomic duplication. Transgenic models with extra copies of the Pmp22 gene
have provided formal proof that overexpression of only this candidate gene is sufficient
to cause peripheral demyelination, onion bulb formation, secondary axonal loss,
and progressive muscle atrophy, the pathological hallmarks of CMT1A. The
transgenic CMT rat with about 1.6-fold PMP22 overexpression exhibits clinical
abnormalities, such as reduced nerve conduction velocity and lower grip
strength that mimic findings in CMT1A patients
From Bolus we have:
A vast majority (over 70%) of CMT cases are due to a DNA
duplication event that consequently leads to abnormal levels of protein
synthesis, which disrupts the normal myelin sheath of peripheral nerves. The
duplicated section of DNA is approximately 1.5 Mb in length and located on
chromosome 17 region p11.2-p12. Within this region is the gene that codes for
Peripheral Myelin Protein 22 (PMP22). As the name suggests, this protein plays
a significant role in the myelin formation among peripheral nerves. The
phenotype of classic CMT is a caused by a “gene dosage effect”. A healthy
individual will have two normal copies of PMP22, one from the mother and one
from the father. Disease is present when this dosage is altered. When
there is a single copy of PMP22 (deletion of one copy), a mild phenotype is
present; Hereditary Neuropathy with Liability to Pressure Palsies (HNPP). When
three copies of PMP22 are present (duplication of one copy), a more severe
phenotype is present; recognized as Charcot Marie Tooth Type 1A (CMT1A). Four
copies of PMP22 (duplication of both copies), though rare, result in the most
severe phenotype Dejerine-Sottas Syndrome (DDS)
The putative simplified
pathway elements of PMP22 are shown below:
It demonstrates the effect on SIVA1 gene product and the
inhibition of apoptosis. As we have stated from NCBI:
This gene encodes a protein with an important role in the
apoptotic (programmed cell death) pathway induced by the CD27 antigen, a member
of the tumor necrosis factor receptor (TFNR) superfamily. The CD27 antigen
cytoplasmic tail binds to the N-terminus of this protein. Two alternatively
spliced transcript variants encoding distinct proteins have been described.
The FGFR appears to have a significant effect on
angiogenesis and growth. This is essential for an aggressive tumor.
As Yang et al state regarding the function of FGFR:
The fibroblast growth factor receptor FGFR1 is
ectopically expressed in prostate carcinoma cells, but its functional
contributions are undefined. … Mice lackingFGFR1 in prostate cells developed
smaller tumors that also included distinct cancer foci still expressing fgfr1
indicating focal escape from gene excision. Tumors with confirmed FGFR1 deletion exhibited increased
foci of early, well-differentiated cancer and phyllodes-type tumors, and tumors
that escaped fgfr1 deletion primarily exhibited a poorly differentiated
phenotype. Consistent with these phenotypes, mice carrying the fgfr1 null
allele survived significantly longer than those without FGFR1 deletion. Most interestingly, all metastases were primarily
negative for the FGFR1 null allele, exhibited high FGFR1 expression and a
neuroendocrine phenotype regardless of FGFR1 status in the primary tumors.
Together, these results suggest a critical and permissive role of ectopic FGFR1
signaling in prostate tumorigenesis and particularly in mechanisms of
metastasis.
Clearly in murine models it is a gene which if uncontrolled
has highly aggressive characteristics.
From Acevedo we have the following pathway characterization
(as modified and simplified:
Note that it reflects on both angiogenesis and
proliferation. The above has been somewhat simplified to highlight the key
elements. The FGFR is activated by the FGF, one of many growth factors.
As Bunz relates there is a direct connection between p53 and
the regulation of cell cycle dynamics. CDKN1A is a direct target of p53
transcriptional transactivation. CDKN1A encodes the gene p21 which is a
universal CDK, cyclin dependent kinase, which regulates multiple cell cycle transitions.
Thus having an activated and over-expressed p21 with excess CDKN1A we have a
significant driver to cell cycle activation and resulting cell proliferation.
p53 associates with a binding motif in the CDKN1A promoter
and significantly increases CDKN1A transcription. Cancer cells which have
impaired p53 also have impaired CDKN1A transcription and thus the cell is
restricted in managing cell cycle response to damaged and incompletely
chromosomes[1].
As Bau et al state:
The protein p21 (Cdkn1a/Waf1/Cip1), encoded by the CDKN1A
locus, is a universal inhibitor of cyclin-dependent kinases (Cdks), which
suggests its widespread role in regulating the cell cycle. The human CDKN1A
gene consists of three exons of 68, 450 and 1600 bp. In normal cells, p21 exists
predominantly in quaternary complexes with cyclins, Cdks, and PCNA to inhibit
the activity of Cdks and control the G1- to S-phase transition. The CDKN1A gene has a p53 transcriptional regulatory
motif and cells lacking functional p53 tumor suppressor protein express very
low levels of p21, suggesting that p53 regulates CDKN1A expression directly.
The expression of p21 induces differentiation of normal and transformed cells,
and the involvement of p21 in terminal differentiation has been observed in
several cell systems. Differential regulation of p21 by p53 and retinoblastoma
has been reported in cellular response to oxidative stress. In addition,
several recent studies suggest a role for p21 in apoptosis. Quercetin-induced
apoptosis in hepatocytes was also associated with the regulation of p21 protein
expression in a p53-independent pathway. [CDKN1A] This gene encodes a potent cyclin-dependent
kinase inhibitor. The encoded protein binds to and inhibits the activity of
cyclin-CDK2 or -CDK4 complexes, and thus functions as a regulator of cell cycle
progression at G1. The expression of this gene is tightly controlled by the
tumor suppressor protein p53, through which this protein mediates the
p53-dependent cell cycle G1 phase arrest in response to a variety of stress
stimuli. This protein can interact with proliferating cell nuclear
antigen (PCNA), a DNA polymerase accessory factor, and plays a regulatory role
in S phase DNA replication and DNA damage repair. This protein was reported to
be specifically cleaved by CASP3-like caspases, which thus leads to a dramatic
activation of CDK2, and may be instrumental in the execution of apoptosis
following caspase activation. Two alternatively spliced variants, which encode
an identical protein, have been reported. Cyclin D is one of the key regulators of the cell cycle.
As Bunz states (Bunz, pp 218-221) the cell cycle goes through several
well-known phases. There are phase specific kinases which are cyclins which are
called that because they were found to increase or decrease in a cyclical
manner as the cell cycle phase progressed. [2]
From Porath and Weinberg we have:
The molecular circuitry of senescence. p53 and Rb are the
main activators of senescence. p53 can activate senescence by activating Rb
through p21 and other unknown proteins, and also, in human cells, can activate
senescence independently of Rb. Rb activates senescence by shutting down the
transcription of E2f target genes. Rb is activated either by p21, or by the
p16INK4a product. p53 activation is achieved by phosphorylation, performed by
the ATM/ATR and Chk1/Chk2 proteins, and by the p19ARF product of the INK4a
locus, which sequesters Mdm2 in the nucleolus. The transcriptional control of
the INK4a products is not fully elucidated, indicated are some of these
regulators.
In the cycles the cyclin binds with a cyclin-dependent kinas
or CDK. The activated cyclin-CDK complex phosphoralates phase specific
substrates. Cyclin D along with CDK4 and CDK6 facilitate the transition through
G1 to the start of S for example. Cyclin E with CDK2 facilitates the transition
from G1 to S. Cyclin A with CDK2 moves through S. Cyclin A/B with CDK1 moves
through G2. Thus activation of Cyclin D is a sign that cell replication has
commenced.
As stated in NCBI[3]:
The protein encoded by
this gene belongs to the highly conserved cyclin family, whose members are
characterized by a dramatic periodicity in protein abundance throughout the
cell cycle. Cyclins function as regulators of CDK kinases. Different cyclins
exhibit distinct expression and degradation patterns which contribute to the
temporal coordination of each mitotic event. This cyclin forms a complex with
and functions as a regulatory subunit of CDK4 or CDK6, whose activity is,
required for cell cycle G1/S transition. This protein has been shown to
interact with tumor suppressor protein Rb and the expression of this gene is
regulated positively by Rb. Mutations, amplification and overexpression of this
gene, which alters cell cycle progression, is observed frequently in a variety
of tumors and may contribute to tumorigenesis
We use the NCI data set for its pathway[4]:
There has been a proliferation of putative gene expression
findings related to a multitude of cancers. We have argued that behind any
putative marker that there should be some causal model reflective of reality.
Our prior focus has been primarily on pathway modifications and specifically
the gene which has been changed in terms of expression that is reflected in
that pathway change.
Let me provide a few observations:
1. The need to determine markers for assessing aggressive
PCa is a critical factor in managing the disease. It is well known that a small
fraction is aggressive but the aggressive forms have devastating morbidity and
mortality effects. The arguments over PSA testing are oftentimes done by those
who have been least affected and even more so least knowledgeable. Having some
definitive test is of help.
2. These three genes arguably cover three of the most
significant factors in PCa. However one must look at them in the context of the
overall networks controlling cells. Namely pathways and causative gene must be
studied. What of methylation and PCa? How is that factors assessed?
3. Sampling genes for the protein levels may be much more
complex than realized. What cells do we sample? What is a metastatic stem cell
has already moved out into distant sites, how is that ascertained? The failure
rates of this approach must be seriously studied.
4. This study appears to be better than many others that
just do some genome wide study and finding a dozen or so genes with some
“correlation”. Here we have some genes with putative causative factors for the
specific disease characteristics.
5. What of the methylation issue and what of the stem cell
issues? How do these fit into this profile?
This work is an excellent step in getting a better hold on
PCa. It will be interesting to see how it progresses.
1.
Acevedo, V., et al, Paths
of FGFR-driven tumorigenesis, [Cell Cycle 8:4, 580-588; 15 February 2009.
2.
Bau, D., et al, Association
of p53 and p21 (CDKN1A/WAF1/CIP1) Polymorphisms with Oral Cancer in Taiwan
Patients, Anticancer Research 27: 1559-1564 (2007).
3.
Beenken, A., et al, The FGF
family: biology, pathophysiology and therapy, Nature Reviews Drug Discovery, volume
8, March 2009, 235.
4.
Bolus, The Function of
Peripheral Myelin Protein 22 (PMP22) in the Context of Tissue Development and
Cellular Differentiation, PhD, Univ Tenn, 2001.
5.
Bunz, F., Principle of
Cancer Genetics, Springer (New York) 2008.
6.
CDKN1A Pathway: http://www.wikipathways.org/img_auth.php/d/df/WP2039_72029.svg
7.
D’Urso, D., et al,
Peripheral Myelin Protein 22 and Protein Zero: a Novel Association in
Peripheral Nervous System Myelin, The Journal of Neuroscience, May 1, 1999,
19(9):3396–3403
8.
Irshad, S., et al, A
Molecular Signature Predictive of Indolent Prostate Cancer, Science
Translational Medicine, 11 September 2013, 5 :( 202): 202ra122.
9.
McGarty, T., Prostate
Cancer System Genomics, DRAFT, January 2013, http://www.telmarc.com/Documents/Books/Prostate%20Cancer%20Systems%20Approach%2003.pdf
10.
Naef, R, A Common Disease
Mechanism for Hereditary Neuropathies Due to Point Mutations in the Peripheral
Myelin Protein 22, PhD, ETH, Zurich, 2000.
11.
Ozen, M., et al, Role of
Fibroblast Growth Factor Receptor Signaling in Prostate Cancer Cell Survival, Journal
of the National Cancer Institute, Vol. 93, No. 23, December 5, 2001.
12.
Porath, I., R. Weinberg,
The signals and pathways activating cellular senescence, The International
Journal of Biochemistry & Cell Biology 37 (2005) 961–976.
13.
Prostate Cancer Pathway, http://www.genome.jp/kegg-bin/show_pathway?hsa05215
, http://cbio.mskcc.org/cancergenomics/prostate/pathways/prostate_cancer_pathways.pdf
14.
Sereda, N., K. Nave, Animal
Models of Charcot-Marie-Tooth Disease Type 1A, NeuroMolecular Medicine, 2006
ISSN0895-8696/06/08:205–216
15.
Taylor, B., et al,
Integrative Genomic Profiling of Human Prostate Cancer
16.
Tong, D., et al, Gene
expression of PMP22 is an independent prognostic factor for disease-free and
overall survival in breast cancer patients, BMC Cancer 2010, 10:682.
17.
Trotman, L., S. Powers, New
Views into the Prostate Cancer Genome, Cancer Cell 18, July 13, 2010.
18.
Whibley, C., et al, p53 polymorphisms:
cancer implications, NATURE Reviews Cancer Vol 9 Feb 2009.
19.
Yang, S., et al, FGFR1 is
Essential for Prostate Cancer Progression and Metastasis, Cancer Research, Published
OnlineFirst April 10, 2013; doi: 10.1158/0008-5472.CAN-12-3274.
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