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Erica Green
Genetics
Dr. Ely
November 4th, 2011
The Effects of Advanced Stages of Cervical Squamous Cell Carcinoma: from
Chromosome to Gene Discoveries
Background:
Cervical cancer is a type of cancer that forms in tissues of the cervix. There are two major
types of cells on the cervix’s surface: squamous and columnar. Most cases of cervical cancer
occur in squamous cells. Cervical cancer progresses from a precancerous lesion known as
cervical dysplasia, then into cervical cancer, which is known to ultimately result in death. Human
papillomavirus (HPV) is the primary etiologic agent for cervical cancer (PubMed Heath 2010).
The discovery of copy number gain/amplification of chromosome 5p:
In addition to presence of HPV, studies have shown that there is a copy-number
gain/amplification of chromosome 5p (p = short arm of chromosome) in advanced stages of
cervical cancer (Heselmeyer et al. 1997). Both Heselmeyer et al. (1997) and Kirchhoff et al.
(1999) observed a recurrent pattern of chromosomal abnormalities on chromosome 5p using
comparative genomic hybridization (CGH). “CGH is a molecular-cytogenetic method for the
analysis of copy number changes (gains and/or losses) in the DNA content that are often in
tumor cells” (Wikipedia 2011a).
Both Heselmeyer et al. (1997) and Kirchhoff et al. (1999) demonstrated that there were
many chromatic abnormalities in SCC tumor samples (Table 1). Though it can be seen that the
greatest frequency of abnormalities was in chromosome 3q, later work (Muralidhar et al. 2007)
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indicated that the copy number gain of chromosome 5p was of particular interest. Therefore, this
summary will focus on the work with chromosome 5.
Table 1:
Heselmeyer et al. (1997) visualized the copy number gain of chromosome 5p through the
generation of an image ratio using CGH technique and multiple fluorophores. Results showed
that chromosome five was green at the top, indicating copy number gain of the short arm of the
chromosome (Figure 1).
Figure 1:
Figure 2:
Karyograms used in both Heselmeyer et al. (1997) and Kirchhoff et al. (1999) showed
gains and losses in chromosome 5 (Figure 2). Lines on the right side represented copy number
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increases and lines on the left represented copy number decreases. Heavy lines symbolized high
copy number. No gain was seen in preinvasive tumor samples (Figure 2), indicating that 5p
chromosomal abnormality is seen in the late stages of cervical squamous cell carcinoma (SCC)
tumors (Kirchhoff et al. 1999).
The discovery of the gene relevant to cervical carcinogenesis in association with the copy
number gain of chromosome 5p:
Because it is difficult to identify 5p driver genes using clinical samples, studies to
identify these genes and their relevance to cervical carcinogenesis often employ the W12
cervical keratinocyte model system (Muralidhar et al. 2007). The W12 cervical keratinocyte
model system is used to “study events associated with spontaneous selection of cervical
keratinocytes containing integrated high-risk (HR) HPV” (Pett et al. 2006). W12 cells are a
nonclonal cell line that originated from a cervical low-grade squamous intraepithelial lesion
(LSIL). “Integration of HR HPV into the squamous cell genome is seen in most SCC cases”
(Dall et al. 2008), and HR HPV integration is thought to be a critical event in cervical
carcinogenesis before chromosomal abnormalities that drive malignant progression occur (Pett et
al. 2004). It is clear that in order to identify genes that are associated with the 5p chromosome
amplification, HR HPV must integrate into the host’s genome. Because HPV is able to integrate
into the genome of W12 cervical keratinocytes, using the W12 model system represented a
“naturally occurring early event” in the development of SCC (Dall et al. 2008).
Muralidhar et al. (2007) used the W12 cervical keratinocyte model system to identify
genes on the 5p chromosome that were relevant to cervical carcinogenesis. They also
demonstrated through microarray analysis that the most upregulated transcript on 5p was the
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major microRNA (miRNA) processing gene, Drosha. Depending on the situation and tissue type,
some miRNAs are thought to have oncogenic activity while others have tumor suppressor
activity (VandenBoom et al. 2008).
Muralidhar et al. (2007) showed that the 5p gain in W12 was associated with
invasiveness. Their version of W12 had copies of integrated HR HPV type 16, the genomic DNA
was tetraploid, and contained multiple isochromosomes of 5p (i5p). “An isochromosome is a
chromosome that has lost one of its arms and replaced
Figure 3:
it with an exact copy of the other arm” (Wikipedia
2011b), meaning that i5p has two short arms rather
than its normal configuration of a short and long arm.
Figure 3 shows three copies of normal chromosome 5
(green arrowheads) and two copies of i5p (yellow
arrowheads).
Another focus of Muralidhar et al. (2007) was “to identify the genes responsible for the
advantage and progression conferred by 5p gain in W12”. Using Affymetic microarrays, they
showed that the most significant increase in expression levels was Drosha, located at 5p13.3
(chromosome 5, short arm, region 1, band 3, and sub-band 3). Results indicated that Drosha
transcripts levels and 5p copy number gain showed “significant” association. Because of the
importance of the Drosha gene in miRNA processing, Muralidhar et al. (2007) wanted to find out
the significance of Drosha expression in miRNA profiles in cervical SCC cell lines and tumor
samples. Their results showed that miRNA mi-R31 showed a higher fold-change in both the cell
lines and tumor samples that overexpressed Drosha in comparison to those cell lines and tumor
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samples that did not overexpress Drosha, “suggesting that miR-31 may contribute to a more
aggressive phenotype in several malignancies” (Muralidhar et al. 2007).
The role of overexpressed Drosha in cervical squamous cell carcinoma (SCC):
Muralidhar et al. (2011) “demonstrated using gene depletion and overexpression
experiments that alteration of Drosha levels in cervical SCC cells caused significant changes in
cell phenotype in vitro and differential expression of key miRNAs”.
Wound-healing assays were done to measure cell migration. The SCC cell lines were
transfected with a pool of four specific small interference RNAs (siRNAs) that would target the
transcripts of interest and knock them down. Results showed that Drosha depletion “significantly
reduced cell migration” (Figure 4), indicating that Drosha played a role in cell migration.
Figure 4:
Muralidhar et al. (2011) then wanted to test for the effects of Drosha depletion on
cervical SCC cell invasion in vitro using a Matrigel matrix invasion-chamber. To visualize those
cells that were considered invaders of the Matrigel matrix, they were stained a crystal violet
color. Results showed that, similar to the observations of the cell migration assay, Drosha
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depletion “significantly decreased invasiveness” SCC cell lines (Figure 5), indicating that Drosha
also played a role in cell invasion.
Figure 5:
Of the 319 miRNAs tested by Muralidhar et al. (2011), 45 miRNAs were shown to be
associated with Drosha levels and oncogenesis. When these miRNAs were analyzed further,
miR-31 was the miRNA that best correlated with Drosha expression levels.
Figure 6:
To confirm the miR-31 and Drosha level correlation, Muralidhar et al. (2011) analyzed
miR-31 expression levels in cervical SCC cell lines. Results in figure 6 showed that those cell
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lines that overexpressed Drosha (CaSki and SW756, for example), also expressed high levels of
miR-31. Similarly, cell lines that did not overexpress Drosha showed low expression levels of
miR-31 (MS751, for example). It was noted by Muralidhar et al. (2011) that miR-31 is known to
be in association with SCC tumors of the cervix. Since Drosha enhances both cell motility and
invasion, it appears that Drosha overexpression may be an important feature of advanced cervical
SCC.
Conclusion:
The discovery of copy number gain/amplification of chromosome 5p (Heselmeyer et al.
1997 and Kirchhoff et al. 1999), led to the idea that overexpression of Drosha was important for
progression in advanced stages of SCC (Muralidhar et al. 2011). This finding may lead to a
better understanding of why cervical cancer caused by some strains of HPV are aggressive while
cervical cancer caused by other strains of HPV are more benign.
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References:
http://www.answers.com/topic/cervical-cancer
PubMed Heath 2010:
http://www.ncbi.nlm.nih.gov/pubmedhealth/PMH0001895/#adam_000893.disease.sympto
ms
http://ghr.nlm.nih.gov/handbook/howgeneswork/genelocation
Heselmeyer K, Macville M, Schro¨ ck E, Blegen H, Hellstro¨m A-C, Shah K, Auer G, Ried T.
1997. Advanced-stage cervical carcinomas are defined by a recurrent pattern of
chromosomal aberrations revealing high genetic instability and a consistent gain of
chromosome arm 3q. Genes Chromosomes Cancer 19:233–240.
Kirchhoff M, Rose H, Petersen BL, et al. Comparative genomic hybridization reveals a recurrent
pattern of chromosomal aberrations in severe dysplasia/carcinoma in situ of the cervix and
in advanced-stage cervical carcinoma. Genes Chromosomes Cancer 1999; 24: 144–150.
Wikipedia 2011a: http://en.wikipedia.org/wiki/Comparative_genomic_hybridization
Muralidhar B, Goldstein LD, Ng G, et al. Global microRNA profiles in cervical squamous cell
carcinoma depend on Drosha expression levels. J Pathol 2007; 212: 368–377.
Pett MR , Herdman MT, Palmer RD, et al. Selection of cervical keratinocytes containing integrated
HPV16 associates with episome loss and an endogenous antiviral response. Proc Natl Acad
Sci U S A 2006; 103: 3822–3827.
Dall KL, Scarpini CG, Roberts I, et al. Characterization of naturally occurring HPV16 integration
sites isolated from cervical keratinocytes under noncompetitive conditions. Cancer Res
2008; 68: 8249–8259.
Pett MR, Alazawi WO, Roberts I, et al. Acquisition of high-level chromosomal instability is
associated with integration of human papillomavirus type 16 in cervical keratinocytes.
Cancer Res 2004; 64: 1359–1368.
Timothy G VandenBoom II, Yiwei Li, Philip A Philip, Fazlul H Sarkar. MicroRNA and Cancer:
Tiny Molecules with Major Implications. Curr Genomics. 2008 April; 9(2): 97–109.
Wikipedia 2011b: http://en.wikipedia.org/wiki/Isochromosome
Muralidhar B, David Winder, Matthew Murray, Roger Palmer, Nuno Barbosa-Morais, Harpreet
Saini,Ian Roberts, Mark Pett and Nicholas Coleman. Functional evidence that Droshna
overexpression in cervical squamous cell carcinoma affects cell phenotype and microRNA
profiles. J Pathol 2011; 224: 496–507.