Orchestration of hepatocellular carcinoma development by diverse liver cancer stem cells
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Hepatocellular carcinoma (HCC) is one of the world’s most aggressive diseases and carries a poor prognosis for patients. Recent evidence suggests that HCC is organized by cancer stem cells (CSCs), which are a subset of cells with stem cell-like features. CSCs are considered a pivotal target for the eradication of cancer, and liver CSCs have been investigated using various stem cell markers. Several hepatic stem/progenitor markers have been shown to be useful for isolating putative CSCs from HCC, although the expression patterns and phenotypic diversity of CSCs purified by these markers remain obscure. Recently, we found that liver CSCs defined by different markers show unique features of tumorigenicity and metastasis, with phenotypes closely associated with committed liver lineages. Furthermore, our data suggest that these distinct CSCs collaborate to orchestrate the tumorigenicity and metastasis of HCC. In this review article, we summarize the recent advances in understanding the pathogenesis and heterogeneity of liver CSCs.
KeywordsHepatocellular carcinoma Cancer stem cell Tumorigenicity Metastasis
Hepatocellular carcinoma (HCC) is the third leading cause of death from cancer worldwide . Its prevalence is mostly attributed to hepatitis B virus or hepatitis C virus infection, and high incidence is observed in Asia and Africa . Increasing occurrences and mortality from HCC have also been observed in most industrialized countries . Therefore, there is an urgent need to develop effective diagnostic and treatment strategies against this disease.
HCC is a heterogeneous disease in terms of morphology, biological behavior, response to treatment, and molecular profile . This heterogeneity has traditionally been explained by the clonal evolution of tumor cells resulting from the progressive accumulation of multiple genetic and epigenetic changes [5, 6]. However, recent studies suggest that its heterogeneity may result from the hierarchical organization of tumor cells by a subset of cells with stem and progenitor cell features known as cancer stem cells (CSCs) . CSCs are highly tumorigenic, metastatic, chemo- and radiotherapy resistant, responsible for tumor relapse after therapy, and able to divide symmetrically or asymmetrically to orchestrate the tumor mass . Therefore, they are considered to be a pivotal target for eradicating HCC . In this review, we summarize recent findings on liver CSCs in terms of heterogeneity and discuss an HCC treatment strategy that targets them.
Cancer cells and stem cells have similar capabilities with respect to self-renewal, limitless division, and the generation of heterogeneous cell populations. The observation of these similarities many years ago led to the proposal that cancer might be a type of abnormal stem cell disease , a concept which has recently been revisited . The generally acknowledged definition of a CSC is a cell within a tumor that possesses the ability to self-renew and to give rise to heterogeneous lineages of cancer cells that comprise tumors in immunodeficient mice . Experimentally, putative CSCs have been isolated using cell surface markers specific for normal stem cells. Stem cell-like features of CSCs have been confirmed by functional in vitro clonogenicity and in vivo tumorigenicity assays. Moreover, accumulating evidence suggests that CSCs play a role in perpetuating various cancers including leukemia and solid tumors [12, 13, 14, 15, 16, 17, 18].
Cell surface markers in liver CSCs
Cell surface markers
Function in CSCs
Calcium channel α2δ1 isoform5
Calcium influx and activation of ERK signaling
ROS-induced DNA damage reduction
STAT3 mediated NANOG regulation
Regulation of redox status through xCT
Activation of Wnt signaling
Heterogeneity of liver CSCs
As described above, various hepatic progenitor markers have been detected in the population of liver CSCs. Purified cell populations using certain stem cell markers show CSC features such as high tumorigenicity, an invasive nature, and chemo- and radiotherapy resistance. However, it is unclear how these markers are expressed in primary HCC tissues or HCC cell lines. It is also unclear whether the CSCs expressing these markers exist in all HCCs or are restricted to a certain subtype. This is an especially important issue when treating HCC patients using molecularly targeted therapy against certain marker-positive CSCs.
In normal fetal livers, hepatoblasts express the biliary markers CK19 and EpCAM, as well as the hepatocyte markers albumin and alpha fetoprotein (AFP) [26, 27, 42, 43]. In addition, numerous studies have demonstrated that hepatic progenitor cells express a variety of markers putatively detected in various ectodermal or mesodermal lineages, including nestin, NCAM, CD34 and c-Kit, CD133, CD90, E-cadherin, and Dlk1 . Hepatoblasts are also considered a heterogeneous population potentially organized in a hierarchical manner with various degrees of differentiation that may be related to their expression of stem cell markers . Indeed, recent studies demonstrated that the characteristics of hepatic progenitors expressing different markers show distinct natures [32, 46]. Normal EpCAM+ and CD90+ oval cells represent two distinct populations: the former expresses classical oval cell markers such as AFP, OV-1, and CK19, and the latter expresses desmin and a-SMA but not AFP, OV-1, or CK19, which indicates that CD90+ populations are more likely to be mesenchymal cells.
We explored the expression patterns of the representative liver CSC markers CD133, CD90, and EpCAM in primary HCC, and found that EpCAM+ and CD90+ CSCs show different gene expression patterns and cell morphology . We further explored the tumorigenic capacity of sorted cells isolated from 15 primary HCCs and 7 liver cancer cell lines . Although the number of samples analyzed was small, tumorigenic EpCAM+, CD133+, or CD90+ CSCs were obtained in 26.6 % (n = 4), 20 % (n = 3), and 13.3 % (n = 2) of 15 HCCs, respectively, when xenotransplanted into NOD/SCID mice.
Interestingly, no EpCAM/CD90 double positive cells were detected in primary HCC, and EpCAM+ and CD90+ cells were distinctive with different tumorigenic/metastatic capacities; that is, EpCAM+ cells were associated with a high tumorigenic capacity and hepatic epithelial stem cell features, while CD90+ cells had a metastatic propensity with mesenchymal vascular endothelial cell features. Importantly, the existence of EpCAM+ cells correlated with high serum AFP values with a tendency for portal vein invasion, whereas the existence of CD90+ cells was associated with a high incidence of distant organ metastasis. Furthermore, CD90+ CSCs abundantly expressed c-Kit and showed chemosensitivity against the c-Kit inhibitor imatinib mesylate, whereas EpCAM+ CSCs showed no such chemosensitivity. These data demonstrate that liver CSCs are not a single entity but exist heterogeneously with distinct CSC marker expression, suggesting that no common liver CSCs expressing particular stem cell markers exist in all HCCs. Our data also indicate that the presence of distinct CSCs is a key determinant of cancer phenotypes in terms of tumorigenicity and metastatic propensity, which may influence the clinical outcome of HCC.
The distinct nature of EpCAM+ and CD90+ liver CSCs raises the question whether these different types of CSCs originate from the same or different type of cells. This question remains elusive, but a recent study investigating three independent cell clones established from the same HCC specimen revealed that these clones maintain common karyotype abnormality but express EpCAM, CD90, and CD133 distinctively with different chemosensitivities against sunitinib , suggesting that distinct liver CSCs expressing different markers may originate from the same type of cells. In terms of liver CSC origin, a recent study demonstrated that acquisition of liver CSC properties is independent of the cell of origin, and liver CSCs can originate from hepatic progenitor cells, hepatoblasts, or adult hepatocytes in mice by forced H-Ras/SV40LT induction and subsequent oncogenic reprogramming . In addition, another study has demonstrated the unexpected plasticity of normal mature hepatocytes to dedifferentiate into progenitor cells in rats , and this type of plasticity has also been reported in breast non-CSCs [50, 51]. Given the cellular plasticity reported in normal and cancer cells described above, it is reasonable to speculate that a similar plasticity may exist in EpCAM+ and CD90+ CSCs that can convert their tumorigenic/metastatic phenotypes and marker expression status. Further studies are required to clarify the role of cell plasticity on heterogeneity of HCC .
Interaction of distinct cell lineages in liver organogenesis and hepatocarcinogenesis
Embryogenesis is characterized by the ordered emergence of an organism made up of a multitude of stem and differentiated cells. Various signaling pathways play crucial roles in the dynamic cell proliferation and motility of organogenesis . For example, in liver organogenesis, liver specification signaling is activated at the ventral endoderm (hepatic endoderm) by the paracrine secretion of fibroblast growth factor (FGF) and bone morphogenic protein (BMP) from the cardiac mesoderm and septum transversum, respectively [53, 54, 55]. Wnt/beta-catenin signaling may also induce hepatic specification . Activation of these signaling pathways results in the formation of the liver bud from the hepatic endoderm. The liver bud is considered to be the earliest developmental stage of liver organogenesis, which coincides with the expression of albumin and AFP .
Embryogenesis and tumorigenesis share similar features including autonomous cell proliferation, motility, homing, dynamic morphologic changes, cellular heterogeneity, and interactions with the microenvironment. Liver cancer development may partially recapitulate fetal liver development in terms of the emergence of cells expressing certain stem cell markers and the activation of signaling pathways during liver development (Fig 1b). Indeed, signaling pathways activated in normal liver development are known to be activated and may be involved in the development and maintenance of liver CSCs. FGF and Wnt signaling has also been implicated in the development of HCC [60, 61, 62, 63], with the latter shown to regulate the self-renewal of hepatoblasts and liver CSCs [20, 31, 64, 65, 66, 67, 68].
Moreover, as observed in the process of normal liver development, the collaboration of CSCs with epithelial or mesenchymal cell features may play an important role in the tumorigenicity and metastasis of HCC (Fig 1b). Our data indicate that EpCAM+ CSCs have no metastatic capacity for distant sites when subcutaneously injected into NOD/SCID mice. However, when CD90+ CSCs were co-injected with EpCAM+ CSCs, EpCAM+ cells could metastasize to the lung, whereas subcutaneous primary tumors showed no difference in size . Furthermore, although imatinib mesylate treatment had little effect on the size of primary subcutaneous tumors, it significantly suppressed lung metastasis potentially through the suppression of CD90+ CSCs.
We found that the effect of CD90+ CSCs on the enhanced cell motility of EpCAM+ cells was mediated, at least in part, through the activation of TGF-β signaling by CD90+ CSCs (Fig 1b) . This suggests that CD90+ cells are not only metastatic to the distant organ but also help the metastasis of CD90− cells, including EpCAM+ cells, which have no distant metastatic capacity of their own. Our data further suggest that imatinib mesylate inhibits distant organ metastasis by suppressing CD90+ metastatic CSCs, albeit with little effect on EpCAM+ tumorigenic epithelial stem-like CSCs, which indicates the importance of EpCAM+ and CD90+ CSC interaction in the process of HCC development, especially in distant organ metastasis. These data suggest the limitations of a treatment strategy targeting only certain CSC marker-positive cells to eradicate HCC, as it is highly possible that marker-positive CSCs exist in each HCC patient with different chemosensitivities against molecularly targeted therapy. Interestingly, we have recently identified that EpCAM+ HCC cell lines show abundant expression of the transcription factor SALL4 and high histone deacetylase activity, and the histone deacetylase inhibitor successfully suppressed proliferation of EpCAM+ HCC cell lines but showed little effect on CD90+ HCC cell lines . Further studies of liver CSC heterogeneity are required to provide better treatment strategies for HCC patients.
There is accumulating evidence that liver CSCs play a key role in the development and perpetuation of HCC, and the importance of targeting CSCs has become clearer. Understanding the diversity of liver CSCs will further the development of personalized medicine targeting patient-specific liver CSCs.
Conflict of interest
The authors declare that they have no conflict of interest.
- 44.Yamashita T, Honda M, Kaneko S. Heterogeneity of liver cancer stem cells. In: Wang XW, Thorgeirsson SS, Grisham JW, editors. Molecular genetics of liver neoplasia. 1st ed. New York: Springer; 2011. p. 301–17.Google Scholar