Pathological malignancies, including cancers in the colon and the liver, are life-threatening because of their high occurrence and lower survival rates. Whereas the five-year (2015-2019) survival rate in Korean cancer patients was 74.3% for colon cancer and 37.7% for liver cancer, the occurrence in 2019 was 279,717 (13% out of the total 2,147,503 occurrences) for colon cancer and 75,261 (3.5%) for liver cancer, as reported (December 2021) by the Korea Central Cancer Registry at the National Cancer Center of Korea. Therefore, both cancer types, with either a high occurrence rate (for colon cancer) or a low survival rate (for liver cancer), can extensively influence the health and economic situations of the population.
Identification of molecular biomarkers for these cancer types would provide clinical benefits, such as developing strategies for earlier diagnosis and therapeutic approaches. Accumulations or mutations of certain oncogenes and tumor-suppressor genes for colorectal cancer are well-known in cases of adenomatous polyposis coli (APC), KRAS proto-oncogene, serine/threonine-protein kinase B-Raf, phosphatidylinositol 3-kinase (PI3K), SMAD family member 4, transforming growth factor β, and tumor protein p53 (TP53) (1). In cases of hepatocellular carcinoma, the protein biomarkers include α‐fetoprotein, β‐catenin, TP53, phosphatase and tensin homolog, axin 1 (AXIN1), and retinoblastoma transcriptional corepressor 1 (2). Thus, both cancer types commonly involve β-catenin and TP53. Moreover, we reported that transmembrane 4 L six family member 5 (TM4SF5) is highly expressed in colon and liver cancer in humans and in mouse models (3, 4). In addition, overexpression of TM4SF5 in hepatocytes or liver can promote non-alcoholic steatohepatitis (NASH) (5) and fibrosis (6). Systemically TM4SF5-overexpressing TgTM4SF5 C57BL/6 mice reveal nonalcoholic steatosis at 1 year old and NASH associated with fibrosis at 1.5 years old (5). Further, the
The loss of APC function by mutations and deletion is well-known for its pivotal role in colorectal carcinogenesis (11). Small dysplastic crypts are evident as pretumoral lesions or adenomas in the intestine of
In this study, we investigated whether a genetic transgenic mouse model with TM4SF5 overexpression together with
To investigate the influence of systemic TM4SF5 overexpression in
Immunohistochemical staining of β-catenin in the tissues revealed that overexpression of TM4SF5 led to the invasive accumulation and increased expression levels of β-catenin in the intestinal tissues of
We next explored how dual expression of TM4SF5 and
Next, we hypothesized that TM4SF5 overexpression without
We found that systemic TM4SF5 overexpression in
The relationship between TM4SF5 and β-catenin in the liver or hepatocytes appears to be in a bidirectional positive linkage. Further, as we have shown in this study, the role of pS9GSK3β in β-catenin stabilization in the intestine/colon was different from that in the liver. TM4SF5 mediates the induction of β-catenin in the liver and intestines (this study), and a previous report revealed that β-catenin mediates TM4SF5 induction in hepatocytes (17). TM4SF5 overexpression in hepatocytes promotes β-catenin transcriptional activity for the growth of a three-dimensional sphere in aqueous conditions; a bidirectional positive cross-talk between CD133 and TM4SF5 transduces a signal to GSK3β/β-catenin activity to induce TM4SF5 (17). For this bidirectional positive cross-talk, TM4SF5 in hepatocytes binds CD133, a well-known biomarker for cancer stem cells (30), including those involved in hepatocellular carcinoma (31). Further, CD133 can trigger Akt activation, pS9GSK3β and β-catenin stabilization, and transcriptional activation of
In conclusion, it is likely that TM4SF5 overexpression in the presence of
Human HT29 and HCT116 (Korean Cell Bank, Seoul National Univ., Korea) were cultured in Roswell Park Memorial Institute 1640 or Dulbecco’s Modified Eagle’s Medium (Welgene Inc., Daegu, Republic of Korea) containing 10% fetal bovine serum (GenDEPOT) and 1% penicillin/streptomycin (GenDEPOT, Barker, TX, USA) at 37°C in 5% CO2. Lipofectamine RNAiMAX or Lipofectamine 3000 was used for the transfections following the manufacturer’s protocols (Thermo Fisher Scientific, Waltham, MA, USA). Cells were checked for mycoplasma every other month, and their identities were confirmed upon receipt from the Korean Cell Bank.
TM4SF5 transgenic C57BL/6 mice were generated (Macrogen, Seoul, Korea), and TM4SF5 expression was confirmed (37). The pcDNA3-hTM4SF5-FLAG plasmid (digested with
Colon (HT29 or HCT116) or liver (SNU449 or Hep3B) cancer cells were grown in culture plates or dishes and harvested at 80% confluency before preparation of whole-cell lysates with modified radioimmunoprecipitation assay buffer (38). Primary antibodies used for immunoblots were as follows: anti-laminins (Abcam, Cambridge, UK), anti-β-catenin, anti-pS473Akt, anti-Akt, anti-c-Myc, (Santa Cruz, Biotechnology, Santa Cruz, CA, USA), anti-Flag, anti-pS9GSK3β, anti-GSK3β, (Cell Signaling Technology. Danvers, MA, USA), anti-pY142β-catenin (ECM Biosciences, Versailles, KY, USA), anti-α-tubulin (Sigma), anti-signal transducer and activator of transcription 3 (STAT3, Chemicon, Rolling Meadows, IL, USA), anti-pY705STAT3 (Millipore, Billerica, MA), anti-HA (BioLegend, San Diego, CA, USA), and anti-fibronectin (Dako Diagnostics, Glostrup, Denmark). Antibodies that detect the human TM4SF5 EC2 (long extracellular loop) sequence or C-terminus sequence (39) or the sequence (117CLID NKWDYHFQETEGAYLRND138) in mouse TM4SF5 were custom designed (Pro-Sci, Poway, CA, USA).
A one-way ANOVA with Dunnett’s or Turkey’s multiple comparison test was performed to determine the significance of differences between two groups. A P-value less than 0.05 was considered significant.
This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2020R1I1A1A01070020 to EMK, NRF-2021R1A6A3A01087300 to JEK, NRF-2018M3A9C8020027, NRF-2020R1A2C3008993, and NRF-2021M3A9D3024752 to JWL).
The authors have no conflicting interests.