Background
Hepatocellular carcinoma (HCC) is the third leading cause of cancer related deaths worldwide [
1]. Evidence suggests that HCC arises as a direct consequence of dysregulated proliferation of hepatic progenitor cells [
2,
3]. Such progenitors, called tumor-initiating stem-like cells (TISCs), have been described in many different malignancies, including HCC, and may account for poor survival and chemotherapy resistance within specific tumors [
4,
5]. Transcriptome analysis of HCC has demonstrated that a progenitor-based (TISC-phenotype) expression profile is associated with a poor prognosis compared with differentiated tumors (hepatocyte-phenotype) [
6-
8]. TISCs exhibit the capacity for rapid tumorsphere formation, enriched stem cell gene expression profile, and efficient tumor initiation
in vivo. Moreover, recent evidence suggests that TISCs have mesenchymal features such as low expression of E-cadherin and high expression of Fibronectin and Zeb1 [
9]. Furthermore, TISCs share multiple gene networks involved in self-renewal (i.e. increased expression of stem cell markers such as NANOG, POU5F1 and BMI-1), drug efflux or resistance to chemotherapy drugs, survival, and pluripotency with embryonic stem cells [
9,
10].
c-Met is a receptor tyrosine kinase that, upon activation by its ligand hepatocyte growth factor (HGF), promotes malignant progression and metastasis in multiple cancers, including HCC [
11,
12]. Interestingly, 40% of HCC cases are c-Met
+, and c-Met expression is associated with a poor prognosis [
11,
13,
14]. Aberrant c-Met activation can occur through multiple mechanisms, including autocrine or paracrine ligand-dependent stimulation, mutational activation or gene amplification [
12]. During development, homozygous deletion of HGF or c-Met is embryonic lethal [
15,
16]. Although HGF/c-Met signaling does not play a role in liver homeostasis during normal physiologic conditions, many studies have demonstrated the important role of HGF in liver regeneration, hepatocyte survival, and tissue remodeling after acute injury. Following c-Met phosphorylation and activation, multiple signaling pathways are involved as downstream targets, such as the PI3K/AKT and MAPK/ERK1/2 pathways [
17,
18].
CD44 is a transmembrane cell adhesion glycoprotein that participates in many cellular processes, including the regulation of cellular growth, survival, differentiation, lymphocyte homing, and motility [
19,
20]. The variety of cellular processes affected by CD44 is likely the result of multiple CD44 isoforms produced by alternative splicing [
21-
23]. CD44s, the smallest (standard) form of CD44 (CD44s) is approximately 80–95 kD and lacks all CD44 variable exons. In breast cancer, cells undergoing EMT exhibit increased CD44 expression and TISC characteristics [
24,
25]. Although, CD44 expression has been described within TISC populations, the isoform responsible for the TISC characteristics remain unclear [
20]. CD44s is the predominant CD44 variant, which is ubiquitously expressed in epithelial tissues, and has recently been proposed to be essential for epithelial-to-mesenchymal transition (EMT) [
26]. Recent studies demonstrate that the RNA binding protein IMP3 stabilizes CD44 mRNA to facilitate cell migration and more importantly, CD44s combined with IMP3 can serve as a biomarker in predicting HCC [
27]. Together, these studies suggest the important role of CD44s in HCC progression.
CD44
+/c-Met
+ cells have been demonstrated to be tumorigenic with stemness characteristics in pancreatic cancer, which suggests a dual role of c-Met and CD44 as regulators of tumor initiation [
28]. More recently, c-Met + inhibitors have been demonstrated to improve overall survival of advanced HCC patients [
12]. Thus, understanding how c-Met elicits its oncogenic activities is important in the development of HCC therapies. Using HCC cell lines, we have previously demonstrated that pharmacologic inhibition of c-Met results in the decreased expression of CD44, which indicates a potential link between CD44s and c-Met activation [
11]. In the current study, we investigate the co-regulation of c-Met and CD44s. Here, we define a specific functional role of CD44s as a tumor-initiating regulator in HCC. Our results demonstrate that c-Met regulates tumor initiation and mesenchymal stemness features through the activation of PI3K/AKT/CD44s cascade. Our study provides insight on how c-Met + HCC may be resistant to standard chemotherapy, implicating the importance of precision medicine to improve overall survival in HCC patients.
Discussion
Hepatocellular carcinoma (HCC), the fifth most common cancer in men and seventh in women, is on the rise in the United States [
34]. Due to the diverse etiologies of HCC, including hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, alcoholic diseases and obesity, and its direct impact on the heterogeneity of the tumor, there are limited treatment options with poor survival [
35]. Sorafenib is the only FDA approved therapy for advanced HCC, however the benefits are modest [
36]. In a randomized clinical trials phase II study, tivantinib, a c-Met inhibitor, has demonstrated to be a promising antitumor agent in c-Met high patients with a median overall survival of more than seven months [
37,
38]. Notably, we have previously demonstrated that the inhibition of c-Met in c-Met + HCC significantly reduces tumor burden [
11]. Together, these studies support the idea that targeted therapy is important for improving the overall survival of HCC patients.
HCC patients with an active c-Met signaling or TISC transcriptome profile have a poor prognosis. In solid tumors, c-Met
+ and CD44
+ cells demonstrate increased TISC gene expression profile, increased tumor-sphere formation, and efficient tumor initiation in limited dilution studies [
5,
28,
39-
42]. In this study, we demonstrate the underlying mechanism of how c-Met elicits its tumorigenic properties through the activation of CD44s to induce a mesenchymal and TISC phenotype. Although the importance of CD44 in tumor progression and TISC populations has been demonstrated, most reports that define TISC populations with CD44 utilize antibodies that recognizes all CD44 isoforms [
20]. However, which CD44 variants are responsible for the TISC phenotypes has yet to be elucidated. In this study, we demonstrate the underlying mechanism of how c-Met elicits its tumorigenic properties through the activation of CD44s in order to induce a mesenchymal and TISC phenotype. Our findings establish for the first time the functional relationship between the CD44 standard variant (CD44s) and c-Met in regulating a TISC phenotype. We confirm that CD44s and c-Met are co-expressed in human HCC by using our own data set [
8,
43]. We discovered a novel regulatory relationship between CD44s and c-Met that controls mesenchymal and TISC phenotype through the PI3K-AKT signaling pathway.
The relationship between c-Met and CD44v6 is well established [
44-
46]. Specifically, c-Met regulates CD44 alternative splicing to promote CD44v6 production through RAS signaling [
47]. In turn, CD44v6 interacts with c-Met by presenting HGF and subsequently sustains RAS signaling to promote cell proliferation [
44,
45,
47]. This positive feedback loop occurs in an HGF-dependent manner. In the MHCC97-H cells both CD44v6 and CD44s isoforms are expressed. In our work, the down-regulation of c-Met leads to a slight change in CD44v6 expression, suggesting that c-Met may also regulate CD44v6. The question arose as to why cancer cells would express both CD44s and CD44v6 isoforms. This different role of CD44 on c-Met is explained by the difference in CD44 isoforms involved [
20]. While CD44v6 amplifies c-Met signaling and cell proliferative through RAS signaling as described by others, our data suggest that c-Met regulates CD44s to promote a mesenchymal and TISC phenotype via the PI3K cascade. While CD44v6 does not play a role in the regulation of a TISC phenotype, it has been demonstrated that CD44v6 is important for cell migration and metastasis by promoting c-Met signaling through ERM (ezrin, radaxin, and moesin) proteins [
21,
45,
48]. By expressing both CD44 isoforms in c-Met + tumors, cancer cells are more likely to be resistant to standard treatment, metastasize, and colonize at distant organ sites. Thus, our current study supports the idea that combination therapy with c-Met inhibitor and CD44 monoclonal antibody may be more effective in anti-tumor activity than c-met inhibition alone. Moreover, the CD44 monoclonal antibody has been demonstrated to be effective in chronic lymphocytic leukemia [
49]. Although the role of CD44v6 in cell migration has been well studied in other solid tumors, its functional role in HCC will need to be further investigated.
In this work, we demonstrate the importance of the c-Met/AKT/CD44s cascade in promoting a TISC phenotype. The down regulation of CD44s significantly decreased tumorsphere formation compared with c-Met shRNA cells. However, CD44s was not able to fully rescue tumorsphere formation after c-Met inhibition, suggesting that c-Met may regulate tumorsphere formation independent of CD44s. The c-Met/HGF signaling cascade is important for morphogenesis during embryonic development and organ regeneration by inducing EMT and can be high-jacked by cancer cells to promote metastasis [
12,
50]. Furthermore, c-Met has been implicated in regulating the stem/progenitor phenotype by transcriptional regulation of stemness factors including NANOG, POU5F1, and Sox2 [
42]. Therefore, it is likely that c-Met, through other mechanisms independent of CD44s, can regulate the TISC and mesenchymal phenotype.
Prior studies have demonstrated that the PI3K/AKT signaling cascades promote a mesenchymal phenotype. Studies have suggested that constitutive PI3K/AKT signaling is required for EMT in squamous cell carcinoma, whereas PI3K/AKT signaling is required for TGFβ induced EMT in breast cancer cells [
51,
52]. Furthermore, TGFβ-induced EMT generates CD44
+/CD24
− TISCs [
25]. Here, we provide evidence consistent with previous findings that the PI3K/AKT signaling is a central pathway for a mesenchymal phenotype through the c-Met/PI3K/AKT/CD44s cascade.
Competing interests
Dr. Rountree declares a small research grant (less than $10,000), which does not include direct salary support, from Bayer Pharmaceuticals. Authors WD, SS, and HD declare no potential conflict of interest.
Authors’ contributions
HD carried out the molecular and in vivo studies and drafted the manuscript. WD assisted in molecular and in vivo studies and manuscript preparation. SS participated in molecular in vitro studies. HD and CBR conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.