- Research article
- Open Access
- Open Peer Review
Contribution of KCTD12 to esophageal squamous cell carcinoma
© The Author(s). 2018
- Received: 19 January 2018
- Accepted: 21 August 2018
- Published: 29 August 2018
It has been shown that the expression of potassium channel tetramerization domain containing 12 (KCTD12) as a regulator of GABAB receptor signaling is reversely associated with gastrointestinal stromal tumors. In present study we examined the probable role of KCTD12 in regulation of several signaling pathways and chromatin remodelers in esophageal squamous cell carcinoma (ESCC).
KCTD12 ectopic expression was done in KYSE30 cell line. Comparative quantitative real time PCR was used to assess the expression of stem cell factors and several factors belonging to the WNT/NOTCH and chromatin remodeling in transfected cells in comparison with non-transfected cells.
We observed that the KCTD12 significantly down regulated expression of NANOG, SOX2, SALL4, KLF4, MAML1, PYGO2, BMI1, BRG1, MSI1, MEIS1, EGFR, DIDO1, ABCC4, ABCG2, and CRIPTO1 in transfected cells in comparison with non-transfected cells. Migration assay showed a significant decrease in cell movement in ectopic expressed cells in comparison with non-transfected cells (p = 0.02). Moreover, KCTD12 significantly decreased the 5FU resistance in transfected cells (p = 0.01).
KCTD12 may exert its inhibitory role in ESCC through the suppression of WNT /NOTCH, stem cell factors, and chromatin remodelers and can be introduced as an efficient therapeutic marker.
- Chromatin remodeling
- Esophageal cancer
- Stem cell
Esophageal cancer is the sixth leading cause of cancer related deaths in the world . Squamous cell carcinoma (ESCC) and adenocarcinoma are the main subtypes of esophageal cancer which are common in developing and developed countries, respectively. ESCC involves more than 95% of esophageal cancers in Asia . ESCC has a hot spot in Asian Esophageal Cancer Belt spreading from the China to Caspian Sea . Despite the novel chemoradiotherapeutic modalities, ESCC has still a five-year survival below 20% because of the late diagnosis in advanced stages of tumor [4, 5]. It has been shown that deregulation of cellular signaling pathways such as WNT, NOTCH, SHH, and BMP is extensively involved in ESCC progression and drug resistance [6–10]. Therefore, targeting such pathways can be efficient in paving the way of targeted therapy in such patients. There is not any reported of a single marker to cover and regulate all of the mentioned pathways in esophageal cancer. Potassium channels regulate a wide spectrum of cellular processes through potassium flow across cell membranes. Cancer constitutes a category of channelopathies disorder highlighting the probable role of potassium channels in cell proliferation. KCTD12 (Potassium Channel Tetramerization Domain Containing 12) is auxiliary subunit of GABA-B receptors which alter the G-protein signaling of the receptors. Its expression is observed in different fetal organs such as cochlea and brain, however, it has low levels of expression in adult tissues . It is involved in stabilizing and up regulation of GABAB receptors . Moreover, KCTD12 can be a prognostic factor of gastrointestinal stromal tumors (GISTs) . KCTD12 facilitates M phase entrance and promote cancer cell proliferation which is done by CDK1 dephosphorylation by KCTD12. Therefore, KCTD12, CDK1, and CDC25B complex play an important role in tumor cell cycle regulation . KCTD12 regulates self-renewal and drug resistance, through the ERK signaling pathway . Colorectal cancer stem cells have also shown a down regulation of KCTD12 which is a differentiation factor in relation with ERK pathway . There is a controversy in KCTD12 function in which, KCTD12 plays as an oncogene in gastrointestinal stromal tumors;  and as a tumor suppressor in colon cancer . KCTD12 is also involved in cell cycle regulation through its interaction with CDK1 and CDC25B . In addition, KCTD 21, 11, and 6, have been reported to regulate the proliferation of medulloblastoma stem cells via the HDAC1 and sonic hedgehog signaling pathway [17, 18]. Epigenetic abnormalities such as changes in signaling pathways and chromatin remodeling have been shown as common characteristics for specific cancers. Notch signaling pathway has been assessed during embryonic development and self-renewal of adult organs. It functions through cell-to-cell contact in the regulation of tissue homeostasis and stem cell maintenance [19, 20]. Deregulation of Notch pathway has been reported in a variety of malignancies [21–23]. Regarding the expression patterns, it can function either oncogenic or tumor suppressive through regulation of cell proliferation, arrest, and differentiation . WNT signaling pathway is also another important regulatory pathway in embryonic development, cell cycle regulation, and cancer . It has been shown that tumor progression is related to the epigenetic and genomic changes . The vital processes such as DNA synthesis, repair, and transcription are regulated by dynamic changes in nucleosome structure which is significantly involved in DNA-binding proteins access to DNA . Therefore, it is inevitable that aberrations in chromatin remodelers are correlated with tumor progression [27, 28]. Homeoproteins are also key components of regulatory pathways which are involved in both organogenesis and oncogenesis. They function as transcription factors in normal tissues through activation or inhibition of their target genes. Therefore aberrant expression of HOX family members can be critical for tumorigenesis, indicating the role of such components in tissue homeostasis [6, 10]. In present study we assessed for the first time a probable correlation between KCTD12 as a K+ ion channel component and other epigenetic processes such as NOTCH/WNT pathways, chromatin remodelers, and HOX genes which are the main oncogenic factors in esophageal cancer. This study was performed to introduce the KCTD12 as a master regulator of chromatin remodeling and signaling pathways during ESCC progression.
Cell culture and transfection
Migration assay and drug resistance
ESCC cells were seeded in 6-well plate, cultured until 90–100% confluent for 24 h, and transfected with KCTD12-pbabe in a serum free media as described above. Monolayers were scratched using a 200 μl pipette tip, and washed with PBS to eliminate the detached cells. Wound closure was checked and images were captured at 0, 24 and 48 h (Optica, Italy). Percentage of wound closure was analyzed using Image J software (1.42 version, national institute of health, USA). All the migration assays were repeated three times. MTT assay was done in tetra-plicate reactions for the 5-Fluorouracil (5-FU) resistance, in which 3 × 104 cells were seeded per well and cultured overnight. The cells were treated with 12.5 μg/ml of 5FU for 48 h. The plates were quantified at an absorbance of 570 nm.
cDNA synthesis and quantitative RT-PCR
Primer sequences and thermal profiles
Sequence (5’to 3’)
Stem cell factors
NOTCH and WNT signaling factors
Chromatin remodeling and HOX factors
Role of KCTD12 in migration and 5FU resistance
We have shown that KCTD12 acts as a tumor suppressor in ESCC through different signaling pathways and chromatin remodeling. Our results showed that KCTD12 decreases the cell migration of ESCC cells through TWIST1 and can be introduced as a therapeutic marker against the EMT process and tumor relapse. Moreover, role of KCTD12 in 5FU resistance introduces that as an efficient marker for the epigenetic targeted therapy of esophageal squamous cell carcinoma.
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
MRA and LL were involved in conception and design. NT and AA were involved in experiments. MM was involved in drafting and analyzing and supervised the project. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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