In class we have learned that DNA can be changed covalently
by the addition of methyl groups to cytosine bases. This alteration is
important in shutting down tumor suppressor genes. This article, “DNA methylation in thyroid tumorigenesis” is about how three tumor suppressor genes,
CASP8, RASSF1, NIS, are being silenced
due to DNA methylation. The clinicians of this study try to identify if maybe
methylation is an early change in thyroid tumorigenesis regardless of the cell
type. Their main goal is to recognize DNA methylation and that can be used as a
biomarker to identify early thyroid cancer.
Background
There are four main types of thyroid cancer, papillary,
follicular, medullary, and anaplastic. For the majority of patients that have
been affected with thyroid cancer and have received proper treatment and
diagnosed early, they have a good prognosis, ”with a 5-year survival rate of
approximately 96%.” Consequently, thyroid cancer has been ignored in the United
States due to this good prognosis. However, those that have been diagnosed late
have a lower “5-year survival rate of under 60%.” As a result, thyroid cancer
continues to be incurable, and the current challenge is to acquire a diagnostic
test that is extremely accurate for early stage thyroid cancer and therapies that
target the cancer for patients that have been diagnosed late. One way to
recognize cancer is by using tumor markers, which can inform the approximate
size of a tumor, the response to the treatment, the possibility of the disease
reoccurring, or notify the progression of the tumor. Tumor markers include
hormones, subgroups of proteins, molecular markers, and epigenetic markers.
Tumor suppressor genes in thyroid cancer have been epigenetic silenced through
DNA methylation. In this study, they observed “promoter hypermethlation in 24
tumor suppressor genes using the methylation-specidic multiplex
ligation-dependent probe amplification (MS-MILPA) assay and in the NIS gene using methylation-specific PCR
(MSP)”
Results
The tumor suppressor genes CASP8 and RASSF1 were the
only genes to be methylated from the assay of the 24 tumor suppressor genes.
Methylation of CASP8 was the most
observed since it seemed to be a early change in “all 5 normal samples, all 3
hyperthyroid samples, and in 3/11 concurrent thyroid cancer with normal thyroid
lesions.” The tumor suppressor gene encodes “Caspase-8 which is a apical
caspase acting in the death receptor-ligand interaction-induced apoptotic
process,” and when hypermethylated, loses its function, which is a contributing
factor in tumorgenesis. Also, methylation of RASSF1 was showed as an early change as well. “It was methylated in
4/5 normal sample, 2/3 hyperthyroid samples and in 4/11 concurrent thyroid
cancer with normal thyroid lesions.” RASSF1
encodes a protein that is comparable to the Ras effector proteins and also
inhibits the accumulation of cyclin D1 in the cycle cell. When methylated of
its CpG-island promoter region, RASSF
loses its function, which plays a key role in tumorgenesis. NIS encodes a protein that is in charge
for the uptake of iodine in tissues. They suggested that the expression of NIS is low; it denotes an early oddity
in the progression of the thyroid cell transformation, since thyroid needs a
small amount of iodine.
Experiment
MS-MLPA is able to observe aberrant promoter methylation in
various cancer genes by using formalin-fixed paraffin embed tissue. It
recognizes changes in methylation and as well as 41 different human DNA
sequence by detecting the HhaI site
in the gene probes of interest. “MS-MLPA panel in the presence of HhaI detects aberrant promoter
hypermethylation by taking advantage of a HhaI
site in the gene probes of interest.
The control gene probes, without a HhaI
site, serve as undigested controls.” Figure 1A. from the MS-MLPA assay illustrates the methylation
of the control, normal thyroid sample, and papillary thyroid cancer cases. When
comparing each section to the control (A), each peak increased significant. For
example, methylation of CASP8 and RASSF1 in case 4-normal thyroid(B) was
increased compared to the control.
Figure 1
A: Normal control DNA sample with 41 individual peaks (red) in the absence of HhaI and 15 separate peaks (blue) in the presence of HhaI. B: Normal thyroid sample with methylation of CASP8 and RASSF1. C: Papillary thyroid cancer (Case 3 - tumor block) with methylation of CDKN2B and RASSF1. D: Papillary thyroid cancer (Case 3 - normal block) with methylation of CASP8 and RASSF1. (PTC – papillary thyroid cancer, A1T – block A1 tumor, A6N – block A6 normal).
For the MSP assay to detect methylation of NIS, they used control unmethylated DNA and control methylated DNA. As a result,
certain cases were methylated and others were not.
Figure 3
Aberrant methylation of NIS
detected by MSP. Lanes 1 & 2: universal methylated and unmethylated
controls. Lanes 3-14 span biopsies for Cases 3, 4 and 13. Note presence
of methylated product in lanes 3, 7 and 9. Note absence of methylated
product in lanes 5, 11 and 13. Lanes 15 & 16: negative control. (A1T
– block A1 tumor, A6N – block A6 normal, B5Met – block B5 metastasis,
Case4-N – normal thyroid, A5T – block A5 tumor, A9N – block A9 normal).
Conclusion
Their data implies that “methylation of CASP8, RASSF1, and NIS maybe
an early change in thyroid tumorigenesis regardless of cell type.” Also that
promoter hypermethylation are promising biomarkers in tumorgenesis and are
capable for molecular targets for cancer detection.
Analysis
1. Link to Class
In class we have studied how genes can loss heterozygosity
and one way is through methylation. CpG methylation is successful in shutting
down the expression of a tumor suppressor gene if it occurs within the promoter
sequence of the gene. In the clinical trial, we see hypermethylation of CASP8, RASSF1, and NIS. As mentioned above, CASP8
encodes for a protein that is involved in apoptosis induced by Fas. When
hypermethylated, CASP8 cannot program
cell death which is one the hallmarks of cancer, resisting cell death. RASSF1
encodes for Ras effector proteins. When hypermethylated, RAS-GTP will
always be turned on and sending signals to the cell and will never be hydrolyze
to RAS-GDP. Also, RASSF1 inhibits the
build up of cyclin D1, which stops the cell cycle. If methylated, then cyclin
D1 is not longer controlled by the extracellular signals by mitogenic factors,
which would lead to uncontrollable cell division and growth. NIS encodes proteins for the uptake in
iodine in tissues. If methylated, then the gene will not be expressed and will
lead to a deficiency of iodine in the thyroid.
2. What evidence supports their goal?
Their main goal was to see if hypermethylation of certain
tumor suppressor genes could be used to detect thyroid tumorigenesis as
biomarkers. They concluded that
their “data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid
tumorigenesis regardless of cell type…hypermethlation are emerging as promising molecular targets for cancer
detection and represent an important tumor-specific marker in tumorigenesis.”
This illustrates that they are not positive in their clinical research and that
hypermethylation maybe be used as a biomarker. The study supposed that
hypermethylation can be used as a biomarker but they are not certain, which
raises skepticism. In order for their goal to be supported by evidence, they
must of gathered a larger sample size and illustrate statistical values instead
of percentages that are not relevant in their study.
3. What could have been done differently?
The certain assay that was conducted in this study might
have been done differently. It would be interesting to see where exactly the
hypermethylation was being expressed on the promoter, if possible. Or an assay
that uses an amplification plot of a tumor with methylated CASP8, RASSF1, and NIS and
unmethylated CASP8, RASSF1, and NIS, so see the expression of that that
certain gene. Also, stronger statistical support of their evidence and
definitely a sample size larger than 30, so their data has less variance. In
addition, it would be interesting to see if methylation of DNA on certain
sequences can correlate to the diagnoses, prognosis, and treatment of a specific
patient.
4. Other therapeutic treatments
Since, certain CpG sequence is frequently methylated in a
few cells and unmethylated in others, this means that methylation of DNA is
reversible. Therefore, there might be enzymes that exist that remove the methyl
groups on the promoter region. However, this has not been discovered yet, but
it would be fascinating is one can invent an artificial enzyme that can remove
the methyl groups. Also, it would be interesting as well if one can discover an
inhibitor that restrains maintenance methylases of attaching methyl groups on the
hemi-methylates. If this were discovered, that would be effective of not
regenerating the same configuration of methyl groups that existed before the replication.
Conclusion
Overall, I believe this study is just the beginning steps of
developing a biomarker that can detect early thyroid cancer in patients and hopefully later
methylation of DNA can inform one’s prognosis, diagnoses, and treatment. I would of liked for them to explain how specifically methylation of
DNA can detect early thyroid tumorigenesis and what diagnostic tests
physicians can run to diagnosed their patients. I
would like to do more research and see if individuals have already conducted a
study with concrete details and significant data of using hypermethylation as
biomarkers. If so, this is truly a promising tumor-specific marker that be can used to detect thyriod cancer in advanced, and help patients that have been diagnosed late.
References
1. "Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National
Library of Medicine. Web. 10 May 2012.
2. Stephen, Josena K., Dhananjay Chitale, Vinod Narra, Kang Chen, Raja
Sawhney, and Maria
Worsham. "DNA Methylation in Thyroid Tumorigenesis." Cancers (2011): 1732-743
3. Weinberg, Robert A. The Biology of Cancer. New York: Garland Science, 2007. Print.
