A Novel Function for KLF4 in Modulating the De-differentiation of EpCAM-/CD133- nonStem Cells into EpCAM+/CD133+ Liver Cancer Stem Cells in HCC Cell Line HuH7.

Zeynep Firtina Karagonlar,Eren Sahin,Kıvılcım Eren Ates,Esra Erdal,Mustafa Karabicici,Tugsan Balli,Canan Celiker,Bilge Karacicek,Nevin Ersoy,Kubra Nur Kaplan,Nese Atabey,Sanem Tercan Avci,Soheil Akbari

doi:10.3390/cells9051198

Zeynep Firtina Karagonlar, Eren Sahin + Show 11 more

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https://doi.org/10.3390/cells9051198

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Abstract

The complex and heterogeneous nature of hepatocellular carcinoma (HCC) hampers the identification of effective therapeutic strategies. Cancer stem cells (CSCs) represent a fraction of cells within tumors with the ability to self-renew and differentiate, and thus significantly contribute to the formation and maintenance of heterogeneous tumor mass. Increasing evidence indicates high plasticity in tumor cells, suggesting that non-CSCs could acquire stem cell properties through de-differentiation or reprogramming processes. In this paper, we reveal KLF4 as a transcription factor that can induce a CSC-like phenotype in non-CSCs through upregulating the EpCAM and E-CAD expression. Our studies indicated that KLF4 could directly bind to the promoter of EpCAM and increase the number of EpCAM+/CD133+ liver cancer stem cells (LCSCs) in the HuH7 HCC cell line. When KLF4 was overexpressed in EpCAM−/CD133− non-stem cells, the expressions of hepatic stem/progenitor cell genes such as CK19, EpCAM and LGR5 were significantly increased. KLF4 overexpressing non-stem cells exhibited greater cell viability upon sorafenib treatment, while the cell migration and invasion capabilities of these cells were suppressed. Importantly, we detected an increased membranous expression and colocalization of β-CAT, E-CAD and EpCAM in the KLF4-overexpressing EpCAM−/CD133− non-stem cells, suggesting that this complex might be required for the cancer stem cell phenotype. Moreover, our in vivo xenograft studies demonstrated that with a KLF4 overexpression, EpCAM−/CD133− non-stem cells attained an in vivo tumor forming ability comparable to EpCAM+/CD133+ LCSCs, and the tumor specimens from KLF4-overexpressing xenografts had increased levels of both the KLF4 and EpCAM proteins. Additionally, we identified a correlation between the KLF4 and EpCAM protein expressions in human HCC tissues independent of the tumor stage and differentiation status. Collectively, our data suggest a novel function for KLF4 in modulating the de-differentiation of tumor cells and the induction of EpCAM+/CD133+ LCSCs in HuH7 HCC cells.

Highlights

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth leading cause of cancer-related deaths worldwide [1,2]
The EpCAM+ /CD133+ liver cancer stem cells (LCSCs) and EpCAM− /CD133− non-stem cells were maintained in a cell culture only up to day 5 before use in the experiments, since they have a tendency to lose their phenotype in a prolonged cell culture (Supplementary Figure S1)
When we compared the stemness-related properties of the two cell populations, EpCAM+ /CD133+ LCSCs showed an increased spheroid formation ability (Figure 1B) and exhibited a greater cell viability upon sorafenib treatment (Figure 1C)

Summary

Introduction

Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the fourth leading cause of cancer-related deaths worldwide [1,2]. Inter- and intra-tumor heterogeneity is a major hallmark of HCC and significantly contributes to treatment failure and chemotherapeutic resistance in HCC patients [3,4,5,6]. Cancer stem cells (CSCs) or tumor-initiating cells represent a group of cells within the tumor with the ability to self-renew and differentiate, and could significantly contribute to the production of heterogeneous differentiated tumor mass [7,8,9,10,11]. The ability to describe the type of intrinsic or extrinsic stimuli that modulate this shift and the transcription factors that regulate the CSCs phenotype are of great importance in targeting these cells in HCC therapy [21]

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