Background
Of the 36 various types of cancers diagnosed world-wide, colorectal cancer (CRC) ranks third among the most frequently occurring cancers and second in terms of cancer mortalities [
1]. Common genetic alterations responsible for the development and progression of CRC include inactivation of the tumor suppressors
Adenomatosis polyposis coli (
APC) (~ 70%) and
TP53 (~ 60%) and mutational activation of
KRAS (~ 40%) [
2‐
7]. For the treatment of CRC, targeted therapy drugs such as bevacizumab and cetuximab, which are inhibitors of angiogenesis and the epidermal growth factor receptor (EGFR) pathway, respectively, have been actively developed [
8]. However, these inhibitors cannot be used for the effective treatment of all CRC patients. Therefore, additional therapeutic strategies for the treatment of CRC must be developed.
Capicua (CIC) is a transcriptional repressor containing a high mobility group (HMG) box domain and a C-terminal motif that are evolutionarily conserved from
Caenorhabditis elegans to humans [
9‐
14]. Through the HMG box and C-terminal domains, CIC recognizes specific octameric DNA sequences (5′-T(G/C)AATG(A/G)(A/G)-3′) to regulate the expression of its target genes [
12,
15,
16]. There are two main isoforms of CIC, the short (CIC-S) and long (CIC-L) form, which are distinguished by their amino-terminal regions [
17,
18]. It is known that CIC is regulated by extracellular signal–regulated kinase (ERK), which is a downstream kinase of the RAS/RAF/MEK signaling cascade. Activation of the MAPK pathway (RAS/RAF/MEK/ERK) results in phosphorylation of CIC, and this ultimately leads to degradation or cytoplasmic localization of CIC [
19‐
21]. CIC controls several essential processes including cell proliferation and tissue patterning in
Drosophila [
13,
22,
23]. In mammals, CIC is required for lung alveolarization, liver homeostasis, brain development and function, and immune cell homeostasis [
24‐
28].
Accumulating evidence indicates that CIC functions as a tumor suppressor in various types of cancers. Previous studies have identified numerous
CIC mutations in patients suffering from various types of cancers, including soft tissue, brain, lung, gastric, prostate, and breast cancers [
9,
29‐
32]. Additionally, chromosomal translocations that generate the CIC-DUX4 chimeric form have been identified in Ewing-like sarcomas [
9,
33‐
35]. Either mutations in or loss of CIC can promote cancer progression via upregulating the expression of
PEA3 group genes (
ETV1/ER81,
ETV4/PEA3, and
ETV5/ERM), the best characterized and reliable CIC target genes [
9,
32,
36,
37]. The PEA3 group factors are known as an oncogenic transcription factor, because the overexpression of these transcription factors promotes cancer cell proliferation and metastasis via activating the transcription of a subset of genes related to control of cell division and migration, such as matrix metalloprotease (MMP), vascular endothelial growth factor (VEGF), and telomerase reverse transcriptase (TERT) [
38]. Several
CIC mutations were found in the CRC patient samples (6 out of 74 samples) [
39], and it is therefore conceivable that CIC may also be involved in the regulation of CRC progression. Regardless, the exact role of CIC in the suppression of CRC progression and the CIC target genes involved in this process remain to be investigated.
In this study, we examined the association of CIC and PEA3 group transcription factors with CRC clinicopathology by conducting analyses of the TCGA dataset and tissue samples derived from CRC patients. We also investigated the molecular basis underlying CIC-mediated regulation of CRC progression through the use of CRC cell lines and mouse xenograft models. Our study identifies the CIC-ETV4 axis as a key molecular module that controls CRC progression.
Materials and methods
Cell culture
HCT116 (ATCC_CCL-247™) and HT29 (ATCC_HTB-38™) colorectal cancer cells were cultured in DMEM (Welgene, Gyeongsan, Republic of Korea) containing 10% FBS (Welgene, Gyeongsan, Republic of Korea) and 1% penicillin/streptomycin (Gibco, MA, USA). Cells were incubated at 37 °C in a 5% CO2 incubator.
Mice
Male BALB/C nude mice (5-week-old) were purchased from OrientBio (Seongnam, Republic of Korea) and were subjected to acclimatization for 1 week. They were then used for the in vivo tumor formation assay. Mice were fed standard rodent chow and water ad libitum and maintained in a specific pathogen-free animal facility under standard 12 h light/12 h dark cycle. All experimental procedures of animal studies followed the guidelines and regulations approved by the POSTECH Institutional Animal Care and Use Committee (IACUC).
Human tissue samples
Human tissue samples were obtained from Soonchunhyang University Hospital (Cheonan, Republic of Korea). The colon tissue samples from 13 patients with CRC were used in this study. Informed consent was obtained from all patients. All procedures were approved by the Soonchunhyang University Hospital Institutional Review Board (SCHCA 2018-07-061-003).
Generation of viruses and stable cell lines
ETV4 shRNA and
CIC sgRNA cassettes were cloned into MSCV-LTRmiR30-PIG (LMP) and lentiCRISPR v2 plasmids, respectively, according to the manufacturer’s manuals. HCT116 and HT29 CRC cells were infected with viral supernatants in the presence of polybrene (Sigma-Aldrich, MO, USA). After 24–48 h, the cells were selected using 2 µg/ml of puromycin (Gibco, MA, USA) for 48 h. For overexpression of CIC-S and ETV4, the cloned pHAGE-FLAG-CIC-S, pHAGE-ETV4, and pHAGE control plasmids were used. The lentivirus production process was described previously [
36]. Viral supernatants were collected at 48 h post-transfection and were used to infect the HCT116 or HT29 cells for 3 sequential days. The cells were used for further biochemical assays as specified in each experiment.
siRNA transfection
ETV4 siRNA (siETV4) was purchased from Bioneer (Daejun, Republic of Korea). The sequences are as follows: siETV4 sense; 5′- GAGGAAUUCAGCUCAGCUUdTdT -3′, and antisense; 5′- AAGCUGAGCUGAAUUCCUCdTdT -3′. One day prior to transfection, 1 × 105 cells were plated in 60 mm plates. After 24 h, the cells were transfected with 120 pmol of siRNA duplexes using Dharmafect 1, according to the manufacturer’s instructions. After 72 h, the cells were used for further biochemical assays as specified in each experiment.
qRT-PCR
Total RNA was extracted using RiboEX (GeneAll, Seoul, Republic of Korea). cDNA was synthesized using a GoScript™ Reverse Transcript kit (Promega, WI, USA), according to the manufacturer’s instructions. SYBR Green PCR Mixture (Toyobo, NY, USA) was used for qRT-PCR analysis. Expression data were acquired using a StepOnePlus™ Real-Time PCR System (Applied Biosystems, CA, USA). Expression levels of each target were calculated using the 2
−ΔΔCt method and were presented as relative mRNA expression. The sequences of primers used for qRT-PCR were previously described [
37].
Cell lysis and immunoblotting
Cells were harvested and lysed in RIPA buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) containing complete protease inhibitor cocktail tablets (Roche, Basel, Switzerland) by sonication. The lysates of CRC patients’ tissue samples were also prepared by sonication in RIPA buffer. The concentration of cell proteins was determined via the BCA assay. Western blot analysis was performed as described previously [
25]. Generation of rabbit polyclonal anti-CIC antibody was previously described [
25]. Anti-ETV4 (10684-1-AP) antibody was purchased from Proteintech (IL, USA). Anti-β-ACTIN (sc-47778) antibody was purchased from Santa-Cruz Biotechnology (TX, USA). HRP-conjugated secondary antibody was purchased from Pierce Thermo Scientific (MA, USA). The western blot images were obtained using ImageQuant LAS 500 (GE Healthcare Life Science, PA, USA).
Cell growth assay
Stably infected cells (7 × 103 cells) were seeded into each well of 24-well plates. The cells were trypsinized and stained with Trypan Blue (Sigma-Aldrich, MO, USA). The number of viable cells was counted using a hemocytometer every day for 4 days. For cell growth assays of siRNA-treated CIC knockout HCT116 or HT29 cells, 7 × 103 cells were seeded into 24-well plates 1 day before transfection, and then siRNAs were transfected using Dharmafect 1 (Dharmacon, CO, USA) and set as day “0”. The cells were trypsinized and stained with Trypan Blue. The number of viable cells was counted using a hemocytometer every day for 4 days.
In vitro migration and invasion assay
A 24-well trans-well plate (8-µm pore size, SPL, Pocheon, Republic of Korea) was used to measure the migratory and invasive abilities of each cell line. For trans-well migration assays, 5 × 104 cells were plated in the top chamber lined with a non-coated membrane. The inserts were cultured in a well of 10% FBS containing media and were incubated for 6 h. They were then removed, washed with PBS, stained with formalin/0.1% crystal violet solution, and analyzed under a ZEISS Axioplan2 microscope. Multiple 5–10 images per insert were acquired, and the average counts were calculated. For invasion assays, chamber inserts were coated with 16 µl/ml of Matrigel (BD Biosciences, MA, USA) with DMEM/F12 media (Gibco, MA, USA) and dried overnight under sterile conditions. Next, 1 × 105 cells were plated in the top chamber. The inserts were cultured in a well of 10% FBS-containing media and incubated for 48 h. The same staining method used in the migration assay was applied.
In vivo tumor growth assay
For xenograft tumor growth assays, control and CIC KO cells (5 × 106 cells) were subcutaneously injected into the posterior flank of 6-week-old male BALB/C nude mice. Seven days after inoculation, the tumor size was measured every week for 12–13 weeks. The tumor volume was calculated as 1/2 × (largest diameter) × (smallest diameter)2.
Tissue microarray and immunohistochemistry
The colorectal cancer tissue microarray (CO2085b) was purchased from Biomax (MD, USA). Formalin-fixed paraffin-embedded specimens were deparaffinized and stained with rabbit polyclonal anti-ETV4 antibody (1:500 dilution). Each sample stained with anti-ETV4 antibody was scored as negative (−), weak (+), or strong (++) according to the staining intensity. These scores were determined independently by two pathologists in a blinded manner. Tissue samples from 9 CRC patients were provided by Soonchumhyang University Hospital (Cheonan, Republic of Korea). Formalin-fixed paraffin-embedded specimens were deparaffinized, and the antigens were retrieved via a citrate-buffered (pH 6.0) solution method. After blocking endogenous peroxidase activity, immunohistochemistry of CIC and ETV4 was performed using a VECTASTAIN Elite ABC HRP Kit (Vector Labs, CA, USA) according to the manufacturer’s instruction. Specimens were stained with home-made rabbit polyclonal anti-CIC antibody (1:500 dilution) [
25] or anti-ETV4 antibody (1:500 dilution). The color reaction was performed using a DAB kit (Vector Labs, CA, USA). Then, the sections were counterstained with Mayer’s hematoxylin, dehydrated, and mounted. Images were acquired under an OLYMPUS BX41 microscope and analyzed by SPOT Basic image capture software.
TCGA database analysis
Gene expression data from colorectal cancer and normal cells (mRNA, normalized RNAseq FPKM-UQ, July 2014) were retrieved from the TCGA database (provisional) using cBioPortal for cancer genomics during the diagnosed period from 1998 to 2013. Gene expression data were available for 453 CRC patients. Expression levels were log2 transformed. Clinical data including the tumor stage were downloaded from the TCGA portal in July 2014. Tumor stages were defined using the latest version of the American Joint Committee on cancer code at the time of diagnosis. Major tumor stages (I, II, III, or IV) were investigated for differences in gene expression. Expression levels of
CIC and
PEA3 group genes after normalization were compared among the tumor stages.
P values were calculated using a Mann–Whitney U test comparing the expression values in the patient samples at each tumor stages. The detailed clinical and pathological characteristics of CRC patients in TCGA database are listed in Additional file
1: Table S2.
Statistical analysis
For statistical analysis, all experiments were performed more than thrice independently. Data are presented as mean ± standard error. The quantitative data were compared between groups using the Student’s t test (two-tailed, two-sample unequal variance). A value of P < 0.05 was considered to be statistically significant.
Discussion
Previous studies have shown that CIC functions as a tumor suppressor in various types of cancers, such as brain, lung, gastric, prostate, and liver cancers [
30‐
32,
36,
37]. In most cases, CIC deficiency promotes cancer progression via derepression of
PEA3 group genes, and the degree of derepression of each member of the
PEA3 group genes is variable among cancer types:
ETV5 is the most significantly and dramatically upregulated in CIC-deficient prostate cancer cells [
36], while
ETV4 is upregulated in liver cancer cells [
37]. Our findings demonstrate that CIC functions as a tumor suppressor in CRC cells and highlight ETV4, among the PEA3 group transcription factors, as a strong promoter of cancer progression and as a critical target of CIC in the context of CRC.
Analyses of the TCGA dataset and tissue samples from CRC patients revealed that CIC expression was more prominently reduced in CRC patients at the protein level than it was at the mRNA level. Somatic mutations of
KRAS occur in over 40% of sporadic CRC, and abnormal activation of mutated KRAS affects the activation of the downstream molecules [
46,
47]. As activation of the RAS/MAPK signaling pathway suppresses CIC activity via degradation or cytoplasmic retention of CIC in
Drosophila melanogaster and mammals [
20,
32,
48], the decreased expression of CIC in samples obtained from CRC patients may result from enhanced activity of MAPKs. Reduced expression of CIC proteins was also observed in tissue samples from patients with other types of cancers, such as prostate and liver cancers [
36,
37]. Therefore, a decrease in CIC levels may be one of the key features occurring in the process of cancer progression in various types of cancers that exhibit hyperactivation of RAS/MAPK signaling.
Alterations in several essential developmental signaling pathways including WNT, NOTCH, and Sonic hedgehog (SHH) are known to be associated with CRC progression [
49] Among these, oncogenic mutations in the WNT pathway genes are prevalent in CRC. Mutations inactivating
APC are found in 70–80% of CRCs, and these are believed to initiate malignant transformation of the colorectal epithelial cells [
7,
49]. However, the majority of APC mutant colon epithelial tumors are benign and never progress to CRC, suggesting that other genetic alterations are required for the development of WNT signaling-mutant colon epithelia into CRC. Given this, it is conceivable that
CIC might be a gene whose loss or inactivating mutations drive CRC development and progression via collaboration with the WNT pathway. Consistent with this, ETV4 stabilizes β-catenin, a key transcription factor mediating WNT signaling, to promote tumor aggressiveness in gastrointestinal stromal tumors [
50]. In future studies, it will be interesting to explore if and how the CIC-ETV4 axis cross-talks with the major signaling pathways such as WNT signaling that are altered in CRC cells.
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