Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, et al. CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest 2010;120: 3326-3339. (Reprinted with permission.)
Cancer stem cells (CSCs) are generally dormant or slowly cycling tumor cells that have the ability to reconstitute tumors. They are thought to be involved in tumor resistance to chemo/radiation therapy and tumor relapse and progression. However, neither their existence nor their identity within many cancers has been well defined. Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13+ cells predominated in the G0 phase of the cell cycle and typically formed cellular clusters in cancer foci. Following treatment, these cells survived and were enriched along the fibrous capsule where liver cancers usually relapse. Mechanistically, CD13 reduced ROS-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In mouse xenograft models, combination of a CD13 inhibitor and the genotoxic chemotherapeutic fluorouracil (5-FU) drastically reduced tumor volume compared with either agent alone. 5-FU inhibited CD90+ proliferating CSCs, some of which produce CD13+ semiquiescent CSCs, while CD13 inhibition suppressed the self-renewing and tumor-initiating ability of dormant CSCs. Therefore, combining a CD13 inhibitor with a ROS-inducing chemo/radiation therapy may improve the treatment of liver cancer.
The race for a successful cancer therapy is probably as old as our knowledge of cancer. Is the checkered flag now waving for CD13? The general concept of cancer stem cells (CSCs) implies that these specialized cells are capable of self-renewal and initiation of tumor growth in immuno-incompetent mice that recapitulates the hierarchical heterogeneity of the initial lesion. CSCs share properties with tissue stem cells in general, such as multiple differentiation and persistence in a dormant or slowly progressing period of the cell cycle. Their low proliferation rate in the resting state is probably the reason for the resilience of CSCs against chemotherapy and radiotherapy, which are more effective against quickly proliferating cells. Although they represent only small portions of tumors, CSCs are thus responsible for the indefinite growth or recurrence of tumors.1, 2 Attempts to isolate CSCs have taken advantage of the ability of a subpopulation of tumor cells, that is, the side population (SP), to exclude the dye Hoechst 33342 in vitro. Because of their high expression of adenosine triphosphate–binding cassette membrane transporters, which export the dye from the cytoplasm, SP cells can be readily separated from other malignant cells taking up the dye. Miscellaneous markers such as CD133, CD90, CD44, OV6, and epithelial cell adhesion molecule are also propagated in primary hepatic tumors; however, none of them have been proved to be unique to stem cells in the context of the liver.3
In this study by Haraguchi et al.,4 the expression of a panel of cell surface markers was investigated in the SP fractions of various liver cancer cell lines. In combination with functional assays such as Hoechst 33342 exclusion and proliferation assays, CD13 was identified as a protein marker correlating with an enrichment of dormant, slow-growing malignant stem cells, with most CD13+ cells resting in the G1/G0 phase of the cell cycle. The putative CSC markers CD133+ and CD90+, in contrast, selected cells in the G1-G2/M phases and the G2/M and S phases, respectively; this is incompatible with the concept of slow-growing stem cells. As a result, CD13+ CSCs more frequently formed tumors in the nonobese diabetic/severe combined immunodeficient mouse model in comparison with the CD13− cell fraction, regardless of the coexpression of CD133 or CD90. Finally, CD13+-enriched cells were also capable of forming spheres in vitro; this is typical of stem cells. Interestingly, within the spheres, CD13-expressing SP cells gave rise to proliferating CD90+ cells, which indicated the transition from dormant CSCs to proliferating and/or differentiating CSCs.
How do these findings fit into the current concept of the liver stem cell? Hepatoblasts, the bipotent liver progenitor cells during embryogenesis, give rise to both hepatocytes and biliary epithelial cells. Although the turnover of these cells is extremely low within a normal liver, they start proliferating after an experimental challenge such as partial hepatectomy or bile duct ligation. Hepatoblasts are widely believed to be the source of putative adult stem cells resting in the canals of Hering of the adult liver. When liver regeneration by hepatocytes is impaired, so-called oval cells—the supposed progeny of the canal of Hering stem cell compartment—emerge and represent the bipotential progenitors of both hepatocytes and biliary cells.5 However, fetal and adult hepatocytes and oval cells fulfill basic criteria of stem cells, such as self-renewal, multiple differentiation potential, and serial transplantability, so they all may be regarded as stem cells of the liver.6-8 Adult hepatocytes and the cells of the canal of Hering stem cell compartment are even slow-growing cells under normal conditions and thus meet the CSC criteria. Indeed, there is evidence in the literature that hepatic progenitor cells may play a role in the development of hepatocellular carcinoma (HCC). Markers such as cytokeratin 7, cytokeratin 8, cytokeratin 18, cytokeratin 19, chromogranin A, and neural cell adhesion molecule have been used to identify such cells in hepatocellular tumors.9, 10 The rat oval cell marker OV6 was detected on undifferentiated cells in mixed tumors containing components of HCC and cholangiocarcinomas; therefore, this type of tumor may originate from a common bipotential progenitor cell.11, 12 Because of the occurrence of various putative stemlike cells in the liver and their participation in hepatic regeneration after injury, it has been proposed that there might be multiple cellular lineages prone to malignant transformation and HCC formation.13 All this substantiates the claim that HCC arises from liver stem cells or even liver CSCs. However, the evidence for their existence or true identity remains circumstantial.
The clinical problem with slow-growing CSCs is their resilience against chemotherapy and radiotherapy; thus, they escape the common therapeutic strategies used to kill the fast-growing majority of tumor cells. In their article, Haraguchi et al.4 suggest that the aminopeptidase CD13 itself might be responsible for the chemoresistance against treatment with doxorubicin or 5′-fluorouracil. After treatment with either of these drugs, the expression of CD13 in the CD13+CD133+ fraction was markedly increased, whereas the expression of CD133 remained largely unchanged; this indicated that CD13 might favor drug resistance. In addition, lower concentrations of reactive oxygen species were observed in the CD13+ cell fraction versus the CD13− cell fraction, and this coincided with a survival advantage after treatment with doxorubicin or 5′-fluorouracil. The use of a neutralizing antibody directed against CD13 or the CD13 inhibitor ubenimex (bestatin) induced apoptosis in HCC cell lines, and this further supported the protective role of CD13 against chemotherapy. After the verification of the expression of CD13 in human HCC samples, the recurrence of tumors was correlated with the detection of CD13-expressing cells around the fibrous capsule. Finally, the inhibition of CD13 in a mouse xenograft model enhanced tumor regression. Taken together, these results strongly imply that CD13 expression and the associated reduction in reactive oxygen species are novel targets for tumor (stem) cell therapy in addition to already familiar adenosine triphosphate–binding cassette transporter expression.
Indeed, the zinc-dependent exopeptidase CD13 (or aminopeptidase N) has been proposed as a chemotherapeutic target for some time because of its multiple functions in cellular processes, which include enzymatic peptide cleavage, tumor cell invasion, differentiation of normal or neoplastic cells, proliferation and apoptosis, and cell migration and adhesion (these are just the most critical functions). Several agents against CD13 have already been developed in order to counter its activities.14 However, with CD13 expressed on a number of cells other than CSCs, any potential drug will exert pleiotropic effects on a variety of biological processes and lack tissue or cell specificity. Currently, there are technologies for resolving this problem; these include targeted drug delivery via the asialoglycoprotein receptor expressed on HCC,15 the use of nanoparticles as anticancer drug carrier systems to release drugs in the acidic endosomal environment after endocytotic uptake,16, 17 and the use of drug-loaded, hydrogel-derived beads in transarterial chemoembolization to occlude the arterial blood supply in targeted tissues and to deliver anticancer drugs in a local fashion to tumors.18 However, even if most of these techniques address a specific organ tumor cell, specificity will still be largely missing, so other cell types in the tumor-bearing organ will be affected as well. Hence, treating HCC with CD13 as a CSC target requires the promotion, maintenance, and recurrence of HCC to be linked to a molecular function of CD13 in the liver CSC, which, as previously outlined, still needs an unequivocal definition. If CD13 and its specific function in CSCs are conserved throughout HCCs, there is a good chance that CSC-specific drug candidates can be created that circumvent the escape from conventional chemotherapy and radiotherapy. For the moment, integrative and comparative genomic analyses of human hepatocellular specimens using microarray technologies19 might be the most suitable tools for allocating specific molecular pathways to novel therapeutic targets such as CD13 and thus improving the specificity and efficacy of potential drugs. Hence, CD13 probably is not the final goal of the race but certainly is an important milestone for which waving the flag is worthwhile.