Significance of IL-8 signaling in CD133 mediated tumor initiation and progression of hepatocellular carcinoma

Abstract

A novel theory in the field of tumor biology postulates that cancer growth is driven by a population of stem-like cells, called tumor-initiating cells (TICs). These TICs are believed to display unique survival mechanisms, and account for failure in therapeutic treatments. It is also believed that, effective treatments against the diseases can only be developed through targeting and eliminating these TICs. We previously identified TIC populations derived from hepatocellular carcinoma (HCC) that are characterized by membrane expression of CD133. As findings from our previous studies were mostly based on HCC cell lines, here, we first identified rare CD133+ subpopulations in freshly resected HCC specimens, but not their non-tumor counterparts. We also found increased CD133 expression to be associated with advanced disease stages, increased recurrence rate and poorer overall survival in HCC patients. Next, we describe a novel mechanism by which these cells mediate tumor growth and angiogenesis by systematic comparison of the gene expression profiles between sorted CD133 liver subpopulations through genome-wide microarray analysis. A significantly dysregulated interleukin-8 (IL-8) signaling network was identified in CD133+ liver TICs isolated from HCC clinical samples and cell lines. IL-8 was found to be overexpressed at both the genomic and proteomic levels in CD133+ cells isolated from HCC cell lines or clinical samples. Functional studies found enhanced IL-8 secretion in CD133+ liver TICs to exhibit a greater ability to self-renew, induce tumor angiogenesis and initiate tumors. In further support of these observations, IL-8 repression in CD133+ liver TICs by knockdown or neutralizing antibody abolished these effects. Subsequent studies of the IL-8 functional network identified neurotensin (NTS) and CXCL1 to be also preferentially expressed in CD133+ liver TICs. Exogenous NTS treatment resulted in concomitant up-regulation of IL-8 and CXCL1 with simultaneous activation of p-ERK1/2 and RAF-1, key components of the MAPK signaling pathway. Enhanced IL-8 secretion by CD133+ TICs can in turn activate an IL-8 positive feedback loop through MAPK signaling. Subsequent studies from CD133 sorted cells found only CD133+ TICs, but not CD133- cells were able to response to exogenous NTS / IL-8 stimulations with concomitant up-regulation of CD133, suggested that the preferential expression of NTS / IL-8 signaling cascade was also important in CD133+ TICs self-renewal and maintenance. Further to its role as a liver TIC marker, CD133 also plays functional roles in conferring TICs properties via regulating NTS / IL-8 / CXCL1 / MAPK signaling. These results suggested that CD133+ liver TICs promote angiogenesis, tumorigenesis and selfrenewal through NTS-induced activation of the IL-8 signaling cascade. In conclusion, our findings had identified rare expressions of CD133 in clinical HCC specimens and hence its prognostic values. We also show for the first time the functional roles of CD133 in conferring tumorigenic potential to liver TICs. The characterization of underlying molecular signaling in CD133+ liver TICs in this study should provide not only a better understanding of the mechanisms regulating this specific population of cells but also novel insights that could allow the development of more effective therapeutic treatments of this disease.