Technological advances and greater understanding of stem cell biology have contributed to the identification and characterization of the tissues and organs involved with these cells.
Two major theories have been proposed to describe the stem cells in cancer: In the stochastic model, every tumor cell has the capacity for tumor initiation and growth, and depends on some random and varying intrinsic factor. In the hierarchical model, CSCs make up only a very small subpopulation of cells within the whole tumor. Uniquely, these cells have a greater capacity for self-renewal, differentiation, and tumorigenicity, and may be more resistant to chemotherapy and radiotherapy.1,2
The CSC hypothesis has received support, since it can explain, in part, the heterogenous nature of hepatocellular carcinoma (HCC), and is one potential mechanistic explanation for its high degree of relapse, metastasis, and resistance to therapies.
Identification of Liver CSCs and Their Role in HCC
Attempts at identifying liver CSC (LCSC) markers in established cell lines have shown a high degree of heterogeneity in expression patterns. Given the variability, it is unlikely that a single, universal LCSC marker for HCC exists. Many cell surface antigens have been identified in LCSCs including CD24, CD44, CD90, CD133, and the epithelial cell adhesion molecule (EpCAM; CD326).1,2
While HCC screening and diagnosis is dependent on imaging and serum alpha-fetoprotein (AFP) levels, their use is limited in some contexts, such as with disease recurrence and postoperative detection. Expression of progenitor stem cell markers may serve as alternative indicators of disease assessment and prognosis.
CD24 is associated with early-onset HCC and one mutation, P170T/T, is more common in patients with hepatitis B virus (HBV) than in those without infection. CD44 expression is associated with recurrence after local ablation therapy and metastasis. Tumors that co-express CD44 with CD90 and CD133 are more aggressive than tumors that express CD90 or CD133 alone. Similar to CD24, CD90 expression correlates with HBV-associated HCC and is linked to poor prognosis. In comparison with patients with CD133-negative tumors, patients with CD133-positive tumors have a shorter overall survival (OS) and higher recurrence rate. EpCam-positive, AFP-positive HCCs are associated with poor prognosis and a high metastatic rate.1
The search for novel CSC markers in HCC is ongoing. The identification of a novel marker that can be easily detected by blood is especially of interest. In a recent study, transcription factor sex-determining region Y-box 9 (SOX9) was identified as a novel CSC marker in HCC, according to results published in Scientific Reports by Takayuki Kawai and colleagues3 from Kyoto University, Japan.
SOX9 plays a vital role in the development of many different tissues and organs, including the heart, lungs, testes, pancreas, and central nervous system. During development, SOX9 helps to keep cells in an undifferentiated state, and is regulated by various cell signaling pathways such as Wnt/beta-catenin, Notch, and transforming growth factor beta (TGFb)/Smad. As with many of the other LCSC markers, SOX9 expression is activated during organogenesis, but then normally switched off in adult hepatocytes. Kawai et al3 sought to determine whether aberrant SOX9 expression was an indicator of CSC in human HCC.
Using flow cytometry, investigators identified greater proportions of SOX9+ cells than SOX9- cells from multiple human HCC cell lines, including Huh7, HLF, PLC/PRF/5, and Hep3B. In Huh7 cells, those that expressed SOX9 demonstrated higher proliferative activity, colony formation, and sphere formation, which are all indicative of a tumorigenic phenotype. Additionally, these cells were more resistant to chemotherapy using 5-fluorouracil (5-FU), with resistance being a hallmark property of CSCs.
SOX9 expression also was linked to epithelial-mesenchymal transition (EMT), another hallmark of tumorigenesis. Epithelial-mesenchymal transition is regulated, in part, by the TGFb/Smad signaling pathway. Compared with the SOX9- population, SOX9+ cells showed greater and more efficient TGFb/Smad signaling as well as greater cell motility. Furthermore, SOX9 activated the Wnt/beta-catenin pathway, which plays a critical role in CSC maintenance.
Additional analysis determined that osteopontin (OPN), one downstream target of Wnt/beta-catenin, was regulated by SOX9 in vitro. Importantly, OPN was highly expressed in the serum of patients with SOX9+ HCC, indicating that OPN could serve as a surrogate marker for SOX9. Furthermore, OPN was a more sensitive indicator of SOX9 expression in primary tumors and was superior to the conventional markers, AFP and protein induced by vitamin K antagonists-II (PIVKA-II). Immunohistochemical analysis of HCC tissues showed that patients with SOX9+ HCC had greater serum OPN levels, greater venous invasion, and poorer recurrence-free survival (RFS). Serum OPN was significantly correlated with regression-free survival.3
Other circulating biomarkers have been identified. A 2015 study by Robin Kelley and colleagues4 with the Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, detected CTCs from whole blood by EpCAM enrichment in metastatic HCC. In the pilot study of 20 patients with HCC, CTC detection was associated with elevated AFP levels (≥400 ng/mL) and vascular invasion. Next-generation sequencing of DNA revealed a number of known HCC mutations.
“The ability to detect and characterize malignant cells in circulation holds enormous promise as a biomarker in HCC, a grim cancer challenged by the inability of conventional noninvasive diagnostic and staging modalities to encompass its great clinical and biological heterogeneity, as well as by a scarcity of tumor tissue available for diagnostic or research purposes,” the authors reported. “With increasingly sensitive and precise technologies for the detection and molecular profiling of rare cells, the genomic interrogation of CTCs may offer a powerful new tool to characterize, and someday to target, the dominant tumor subclones responsible for treatment resistance or metastatic progression.”
Others have examined the presence of LCSC markers in blood and their correlation with HCC prognosis. In one study by Fan et al,5 patients with >0.01% circulating CD45+CD90+CD44+ LCSCs had a lower 2-year RFS rate (23% vs 64%) and OS rate (59% vs 94%) than patients with <0.01% circulating LCSCs.
Isolation of CTCs from circulation is critically important. Since taking tumor biopsies is not the standard of care for HCC, serum analysis can function as a noninvasive method for determining the presence of metastatic tumor tissue in real time and allow for assessment of molecular profiling using a “liquid biopsy.” This is particularly vital in HCC, given the associated risks with tumor biopsy and the acceptance of radiographic diagnosis in the absence of tissue confirmation, which also contributes to a lack of untreated tumor samples for research efforts.
Despite the large degree of marker heterogeneity among HCCs, therapeutic targeting of LCSC surface markers and signaling pathways that regulate their expression could be an efficacious approach, given their role in tumor progression, recurrence, and metastasis. Although mostly in preclinical development, some studies have shown promise for strategies that target CSCs.
Therapeutic Strategies Targeting CSCs in HCC
CSCs are inherently resistant to chemotherapy and ionizing radiation, enhancing the need for alternative and novel approaches that interfere with the self-renewal and survival characteristics of CSCs. Such therapies include antibody therapy, epigenetic therapy, and molecular-targeted therapy.
Antibody therapy using monoclonal antibodies (mAbs) against CSC-specific antigens has shown some efficacy in vitro and in vivo. In a mouse xenograft model, the combination of a CD13 antibody bound to 5-FU significantly reduced tumor volume compared with single treatment with either agent.6 A human CD133 mAb conjugated to monomethyl auristatin F (MMAF) inhibited the growth of hepatocellular and gastric cancer cell lines in vitro and in a xenograft model.7
Two investigational compounds, napabucasin (BBI-608) and amcasertib (BBI-503), have emerged as potential CSC inhibitors and are currently being investigated in clinical trials. Napabucasin is a cancer stemness inhibitor that was developed to inhibit CSC signaling by targeting the STAT3 pathway. Amcasertib is an inhibitor of multiple serine-threonine stemness kinases, which inhibit Nano and other cancer stemness pathways.8 Stemness is a term that was initially used to describe stem cell gene expression, but is now used to express characteristics shared by both embryonic and adult stem cells.
Stemness is measured by a cell’s ability to form spheres in culture. Hypermalignant CSCs, or stemness-high CSCs, are highly tumorigenic and metastatic, and are resistant to conventional chemotherapy and radiation. These traditional treatment modalities have been shown to induce stemness genes in cancers, having the negative effective of converting stemness-low CSCs to stemness-high CSCs. Some hypothesize that this is a significant driving factor of relapse in HCC.9
Stemness-high CSCs have traditionally been difficult to target effectively due to increased DNA repair ability, overexpression of drug efflux pumps, and the activation of prosurvival and antiapoptotic signaling pathways. An early preclinical study demonstrated that napabucasin inhibited sphere development in stemness-high CSCs and downregulated their stemness gene expression. Additionally, napabucasin inhibited metastasis in a spontaneous liver metastasis model of colorectal cancer.9
Ongoing clinical trials will continue to investigate napabucasin and amcasertib in HCC and other tumor types. In a phase I/II open label, 3-arm, dose-escalation study, napabucasin or BBI-503 in combination with sorafenib will be evaluated in patients with advanced HCC who have not received prior systemic chemotherapy (NCT02279719). A phase II, open-label, multicenter study will investigate amcasertib in patients with advanced hepatobiliary cancer, including HCC and cholangiocarcinoma, who have exhausted all currently approved standard anticancer therapies (NCT02232633). Lastly, in a phase I open-label study, napabucasin in combination with sorafenib will be investigated in Japanese patients with advanced HCC (NCT02358395).
Napabucasin and amcasertib are being intensely investigated in several other tumor types. The results of multiple early clinical trials were recently presented at the 2016 ASCO Annual Meeting. The results of these trials have been promising.8
“The napabucasin data at ASCO this year are exciting because they underscore the potential of cancer stem cell pathway inhibition across multiple tumor types,” commented Tanios Bekaii-Saab, MD, FACP, co-leader of the Gastrointestinal Cancer Program at the Mayo Clinic Cancer Center. “Additionally, these data demonstrate the potential to combine napabucasin with current standards of care to address unmet needs, including treatment in patients who have had multiple lines of prior therapy.”8
With regard to the expansion of clinical studies with the stemness inhibitor, Chiang J. Li, MD, FACP, President, CEO, and Chief Medical Officer of Boston Biomedical, and the head of Global Oncology for Sumitomo Dainippon Pharma Group, said in a press release, “These studies continue to show napabucasin’s safety and early efficacy across doses and in combination with a variety of established agents. We plan to apply these findings as we advance and expand our clinical development program for this first-in-category cancer stemness inhibitor.”10
Other therapies are also showing promise. In a recent study, Ogawa et al11 utilized VB4-845, an agent consisting of an anti-EpCAM antibody linked to Pseudomonas aeruginosa Exotoxin A. In EpCAM-expressing HCC cell lines, VB4-845 suppressed sphere formation.
Additionally, in combination with 5-FU, VB4-845 significantly reduced tumor growth in a xenograft model. Further studies in preclinical and clinical trials are needed to validate the clinical utility of this approach.
Additional novel alternative strategies involving CSCs have hit some roadblocks. Umbilical cord mesenchymal stem cells (UCMSCs) initially held promise as novel delivery vehicles for therapeutic agents. However, in a recent report, Chang Liu et al12 unveiled potential pro- and antitumorigenic properties of UCMSCs.
Following the birth of a child, UCMSCs can be readily collected and cultured from the umbilical cord. Uniquely, this type of stem cell exhibits a high proliferation rate, wide multipotency, and hypoimmunogenicity.13Generally, the capacity of MSCs to migrate to sites of inflammation or injury is one characteristic that makes them especially attractive as drug carriers. However, studies have demonstrated that both oncogenic and tumor-suppressive properties are present in MSCs, and depend highly on the stem cell origin and tumor model used.14
While most in vitro studies of UCMSCs have used 2-dimensional tissue culture models, Liu and colleagues used a 3-dimensional, co-culture system of UCMSCs and HCC cells to more closely mimic in vivo conditions. Cell growth, CSC characteristics, drug resistance, and metastasis were evaluated.
Co-culture of the HCC cell line, HCCLM3, with UCMSCs enhanced many tumorigenic properties. These included an upregulation of matrix metalloproteinase (MMP) and EMT-related genes (N-cadherin and vimentin), as well as increased migration capacity. These characteristics were reversed in the presence of the TGFb receptor inhibitor SB431542, indicating the importance of this pathway in EMT and HCC metastasis.
To conclude, HCC is a heterogenous disease that is difficult to treat with traditional therapies. The cancer stem cell hypothesis is one explanation for therapy resistance observed in advanced disease. Advances in detection methods have identified several markers of liver CSCs and markers that are suitable for noninvasive, liquid biopsy. Ongoing clinical trials will assess inhibitors to stemness factors in combination with sorafenib in patients with advanced HCC. Although UCMSCs have potential as drug carriers, studies have indicated that they exhibit both pro- and antitumorigenic properties, warranting further investigation.
“Understanding the mechanisms that underlie CSC maintenance to combat cancer is essential,” commented Matilde E. Lleonart, PhD, of the Vall d'Hebron Research Institute in Barcelona, Spain. “Determining the mechanisms that govern radio/chemoresistance in CSCs may illuminate the origins of this complex process and improve the treatments of cancer.”
Bergfeld SA, DeClerck YA. Bone marrow-derived mesenchymal stem cells and the tumor microenvironment. Cancer Metastasis Rev. 2010;29(2):249-261.