Gene expression profiling and pathway analyses
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Gene expression profiling and pathway analyses following the
targeting of HH signaling by GANT61 in human colon carcinoma
cell lines
Leanne Woods
Dr.Ting Shi, Jennifer DeVecchio, Dr. Tanvi Mazumdar, Dr. Akwasi Agyeman,
Dr. Janet A Houghton
Department of Cancer Biology
Lerner Research Institute
Cleveland Clinic
Gene expression profiling and pathway analyses
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Abstract
The Hedgehog (Hh) signaling pathway is implicated in the development of
colorectal cancer. Many cancers display elevated Hh signaling activity; this
persistent activation promotes cellular proliferation.
GANT61 is a newly discovered small molecule inhibitor that is able to block Hh
signaling by inhibiting the Gli1 and Gli2 transcription factors. By inhibiting Gli
activity, GANT61 has significant antiproliferative effects that can potentially be
used to treat cancers that display a noted increase in Hh signaling activity.
Microarray gene expression profiling has become a powerful tool for elucidating
genetic interactions, understanding signal transduction pathways, and identifying
genetic biomarkers correlated with disease classification and chemotherapeutic
treatment responses. The goal of this study was to identify downstream target
genes and pathways specific to the regulation of the Hh signaling pathway in colon
carcinoma. Illumina GenomeStudio was utilized to perform microarray gene
expression profiling of the most significantly modulated 18,401 genes following
the GANT61 induction of HT29 and GC3/c1 colon cancer cell lines. We also
investigated the modulation of canonical pathways induced by GANT61 using
Ingenuity Pathway Analysis. The current analysis revealed commonly upregulated genes, commonly down-regulated genes, and unique gene expressions in
GANT61-treated HT29 and GC3/c1 cell lines. Results from both cell lines
indicated that the inhibition of the Hh signaling pathway using GANT61, induced
diverse gene expression and pathway modulation, especially genes and pathways
which control cell cycle progression and cellular apoptosis. These studies verified
the important role of the Hh signaling pathway in the maintenance and survival of
colon cancer cells.
Gene expression profiling and pathway analyses
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Gene expression profiling and pathway analyses in GANT61-treated HT29 and GC3/c1
human colon carcinoma cell lines
Studies on chemoresistant cancer cell lines validated the activation of Hh pathway which
is an essential factor of metastatic cancer cells and cancer cell progenitors[1]. In general, Hh
signaling plays a pivotal role in a number of normal cellular processes including embryogenesis,
patterning of vertebrate organ structures, adult tissue homeostasis maintenance, tissue repair,
cellular proliferation, and cell survival [2-10]. Human Hh signaling pathway contributes greatly
to juvenile growth and development, but is largely absent in normal adult tissues. Therefore, the
adult activation of the canonical Hh signaling cascade is aberrant. This signaling becomes reactivated in many types of cancers and uncontrolled Hh pathway activation can contribute to
cancer development [11]. While this much is presently known, genomic approaches to shed light
on the role of Hh in colon cancer are lacking and regulatory genes participating in the process
remain incompletely characterized [6].
Completely effective cancer treatment specific to colon malignancies have not been
developed because of drug resistance. This resistance is a major limitation in the clinical efficacy
of anticancer chemotherapy because it is associated with the activation of oncogenic pathways
inducing survival pathways and convergent inhibitors of apoptosis [1]. Because of this, therapies
that target pathway inhibition offer an efficient treatment opportunity.
DNA microarray
technology is leading to the development of new cancer treatments that are tailored to the
molecular characteristics and phenotype of the tumor and the patient. This will allow for
increased tumor response rates and decreased toxicity to the patient [12].
Gene expression profiling and pathway analyses
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GANT61 is currently under investigation as a potential chemotherapeutic agent that
serves as a downstream inhibitor of Hh signaling because of its ability to block both Gli1- and
Gli2-induced transcription. These Gli proteins are transcription factors that constitute the
essential and ultimate effectors of the Hh signaling pathway. Gli1 and Gli2 possess distinct as
well as overlapping functions to activate and repress cellular activities which differ in varying
tissue and cell types [13].
However, their specific roles in regulating Hh-driven cellular
proliferation, survival, and apoptosis require a deeper investigation.
GANT61 researchers have previously studied treatment effects on cancerous tumors
utilizing the mouse model. As demonstrated in vivo, GANT61 was able to reduce tumor growth
in nude mice until no tumors were palpable. GANT61 is a potentially effective cancer treatment
under investigation because of its lack of adverse side effects such as weight loss, ulcerations,
and general non-well being [14].
The goals of the current study were to identify downstream target genes and pathways
specific to the Hh in colon carcinoma as well as to elucidate diverse gene expression and
pathway modulation in HT29 and GC3/c1 colon cancer cell lines that were treated with
GANT61. The expectation was that genes and pathways involving cell cycle progression and
apoptosis would be identified.
Gene expression profiling and pathway analyses
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Materials and Methods
Human colon carcinoma cell lines
HT29 was purchased from ATCC (Manassas, VA). GC3/c1 was established in culture from a
human colon adenocarcinoma xenograft model by the Houghton Lab at Lerner Research
Institute, Cleveland Clinic . Both HT29 and GC3/c1 cell lines express mutant p53 alleles. Cell
lines were preserved in folate-free RPMI 1640 medium containing 10% dFBS and 80nM [6RS]
5-methyltetrahydrofolate.
Flow cytometry
Flow cytometry was utilized to analyze the effects of inhibition of Hh signaling on phase
distribution of cells within the cell cycle. HT29 and GC3/c1 cancer cells were plated in six-well
plates at a density of 100,000 cells per well. Cells were treated with GANT61 (20M; Enzo Life
Sciences, Germany) or vehicle control (0.2% DSMO) in duplicate for 24 hours following
overnight attachment.
This was followed by washing with PBS, trypsinization, and
centrifugation. The cells were fixed at 22oC with 70% ethanol, stored at -20o C, centrifuged at
200 x g for 5 minutes, and washed in PBS. The cells were then suspended in PBS. Low
molecular weight DNA was extracted, centrifuged, and resuspended in a DNA staining solution
that contained 50g of propidium iodide and 2mg of DNAse-free RNAse.
A 30 minute
incubation period followed in the dark at RT. A FACSCalibur flow cytometer was used to
analyze the distribution of cells through the cell cycle and CellQuest software analyzed the data.
See Figure 1.
Gene expression profiling and pathway analyses
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Venn Diagram
A Venn Diagram was created to summarize the Differentially Expressed Genes (DEGs) in
GANT61-treated HT29 and GC3/c1 cells. Illumina Human-ref8 V3.0 Bead-Chip array was used
to determine changes in gene expression. The DEGs consisted of genes with a False Discovery
Rate (FDR)-adjusted p-value <0.001 and fold change 1.5. See Figure 2.
Heatmap
Matlab (Mathworks) was utilized to generate a heatmap to compare the fold change of induced
DEGs in HT29 and GC3/c1. Two steps were taken to select the genes in comparison. First, the
genes with differential expression p-value <0.001 were selected. Second, the up-regulated genes
with fold change 4 and the down-regulated genes with fold change -3 were chosen in each cell
line as these were the maximum extents of positive and negative fold change. Specific genes
were classified into categories defined by roles such as G1/S transition, S-phase progression,
DNA replication or repair, and regulation of the G2 or M-phase transitions. See figure 5.
Ingenuity Pathway Analysis (IPA)
Differentially expressed genes were uploaded and mapped in the IPA database for canonical
pathway analysis (Ingenuity Systems, Mountain View, CA). Up-regulated and down-regulated
gene datasets were analyzed in specified pathways involving cell cycle progression at G1/S and
G2/M. See Figure 4.
Gene expression profiling and pathway analyses
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qRT-PCR
HT29 and GC3/c1 cells were either untreated with 0.2% DSMO or treated with 20 M GANT61
for either 0 hours, 16 hours, 24 hours, 38 hours, or 48 hours at 37oC. Reverse transcription was
accomplished using 1g total RNA to prepare cDNA.
iScript Select cDNA Synthesis Kit
(Biorad) Reverse Transcription System was used for this process. Analysis was performed with
an Applied Biosystems 7500 PCR Detection System (Applied Biosystems Inc.). Primer-BLAST,
NCBI software was used to perform amplifications primed by pairs of chemically synthesized
18- to 24- mer oligonucleotides. Target amplicons of 50-200 bp were generated. The qRT/PCR
reaction conditions consisted of activation for 10 minutes at 95oC with 40 cycles of denaturation
for 15 seconds at 95oC, primer annealing and extension for 1 minute at 60C and ramping back
to 95oC. The extent of amplification at the end of each cycle was able to be determined with
fluorescence readings. Using the comparative CT method, mRNA expression levels of target
genes were normalized to the expression of glyceraldehydes phosphate dehydrogenase (GAPDH)
and quantified. See Figure 3.
Gene expression profiling and pathway analyses
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Results
Flow cytometry: cell cycle arrest
GANT61 was shown to induce the accumulation of cells at G1/S in HT29 and GC3/c1
cells. Both cell lines demonstrated the G1 cell accumulation 24 hours after treatment with
GANT61. These results support the evidence of cell cycle phase arrest at G1 that leads to
apoptosis.
Figure 1
Gene expression profiling and pathway analyses
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Venn Diagram: profile of Differentially Expressed Genes (DEGs)
1,368 genes were differentially expressed in HT29 cells and 1,002 genes were
differentially expressed in GC3/c1 cells. The Venn Diagram provides a quantitative visual
account of both common and unique DEGs to HT29 and GC3/c1. 755/558 genes were upregulated and 613/444 were down-regulated. 763 genes were unique to HT29 and 397 genes
were unique to GC3/c1. In HT29, 459 (60%) were up-regulated and 304 (40%) were downregulated. Similarly, in GC3/c1, 262 (66%) were up-regulated and 135 (34%) were downregulated. 605 genes were common to both cell lines; this represented 3.4% of the total genes.
Of these, 296 (49%) were up-regulated while 309 (51%) were down-regulated.
Figure 2
Gene expression profiling and pathway analyses
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qRT-PCR: validation of gene expression
qRT-PCR was performed for 0 hours, 16 hours, 24 hours, 38 hours, and 48 hours after
GANT61-treated HT29 and GC3/c1 cDNA.
GAPDH was utilized for qRT-PCR data
normalization. This procedure revealed a down-regulation at G1/S of E2F2, CCNE2, CDC25A,
and CDK2 and an up-regulation at G1/S of CDKN1A and CDKN2B. At G2/M, there was a
down-regulation of CCNA2, CDC25C, CCNB2, and CDK1.
qRT was able to accurately
confirm changes in gene expression in HT29 and GC3/c1 cancer cells following treatment with
GANT61.
Figure 3
Gene expression profiling and pathway analyses
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Ingenuity Pathway Analysis (IPA): modulation of canonical signaling pathways
Genes that demonstrated significant expression following GANT61 treatment were
assigned to different canonical signaling pathways and subsequently analyzed using IPA. The pvalue for the 15 most significantly altered canonical pathways in HT29 ranged from 2.045 to
9.025. The p-value range for GC3/c1 was 2.32 to 7.509.
12 of the 15 pathways involving genes significantly down-regulated were common to
both cancer cell lines (indicated by blue lines in Figure 4). The 3 common pathways that
exhibited the greatest down-regulated differential expression included genes involved in DNA
damage response, cell cycle checkpoint control, and mitosis (indicated by red lines in Figure 4).
The other down-regulated pathways involved the G1/S and G2/M DNA damage checkpoints,
DNA precursor metabolism, and cell signaling. Some cell signaling involved different pathways
that were unique to cancers, in general, and colon cancer.
8 of the 15 pathways involving genes significantly up-regulated were common to both
cancer cell lines (indicated by blue lines in Figure 4). The up-regulated gene expression, in
general, demonstrated more diversity.
The common up-regulated pathways were mostly
involved in metabolism in roles dealing with steroids, pyruvate, glycolysis, glutathione, and
glycerolipid. The up-regulated gene expression was not directly related to cellular proliferation
control.
Gene expression profiling and pathway analyses
Figure 4
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Heatmap: up-regulated genes (red); down-regulated genes (green); asterisks (*) denote genes with
specific roles in G1/S-, G2-, or M-phase transitions as well as DNA replication or repair Figure 5
Gene expression profiling and pathway analyses
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Discussion
The Hh signaling pathway is growing as an important area of research because of its
activating role in the development of many human cancers. This activation comes as a result of
mutations in the genes that regulate canonical Hh signaling [4]. Hh signaling is transcriptionally
activated by Gli1 and Gli2 and is significantly influenced by the cellular context of gene
expression. There are differing levels of activation dependent upon the type of tissue or cell
origin. The inhibition of Hh pathway activity has been shown to prevent cancer growth [15-17].
In this current study, GANT61 was able to inhibit the transcriptional function of both
Gli1 and Gli2.
Both HT29 and GC3/c1 colon cancer cell lines accumulate in G1-phase
following GANT61 treatment with a subsequent decrease in following cell cycle phases which
suggested induction of a G1/S checkpoint. The signals that promote this cellular accumulation
lead to the repression of genes that regulate further cell cycle progression.
Following statistical analyses, 1,368 genes in HT29 and 1,002 genes in GC3/c1 were
determined to be significantly modulated with a p-value 0.001 and fold change 1.5 when
treated with GANT61. 296 genes were commonly up-regulated in both cell lines and 309 genes
were commonly down-regulated in both cell lines. Some of the important down-regulated genes
identified at G1/S included E2F2, CCNE2, CDC25A, and CDK2. The important up-regulated
genes at G1/S were CDKN1A and CDKN2B. At G2/M, the significant down-regulated genes
were CCNA2, CDC25C, CCNB2, and CDK1. In addition, novel genes involved in DNA
damage response, stress response, and DNA replication and repair were identified following Hh
signaling inhibition.
Gene expression profiling and pathway analyses
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Regarding the canonical pathways, the down-regulated differentially expressed
pathways included genes involved in DNA damage response, cell cycle checkpoint
control, and mitosis, G1/S and G2/M DNA damage checkpoints, DNA precursor metabolism,
and cell signaling. The up-regulated gene expression, in general, demonstrated more diversity
and included genes involved in metabolism. These metabolic roles were specific to steroids,
pyruvate, glycolysis, glutathione, and glycerolipid.
In summary, a comparison of gene expression profiles was conducted in HT29 and
GC3/c1 human colon carcinoma cell lines while using GANT61 to target the function of Gli1
and Gli2, transcriptional regulators of Hh signaling.
As evidenced from flow cytometric
analysis, cDNA microarray gene profiling, and qRT-PCR, GANT61-treated cells accumulate at
the G1/S cell cycle boundary. Genes that regulate cell cycle transition through G1/S, cell cycle
progression, and cellular proliferation are closely involved with the inhibition of the
transcriptional regulation of Hh signaling in human colon carcinoma cells.
Gene expression profiling and pathway analyses
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