Colon cancer is the third most commonly diagnosed type of cancer in both men and women fifty years of age and older and is the second most common cause of cancer-related death. Most available therapies effectively kill bulk populations of cancer cells, but tumors may regenerate from therapy-resistant minority populations, which may coincide with cancer stem cells. Cancer stem cells capable of self-renewal and differentiation, which are responsible for tumor growth, have been identified in human hematological malignancies and several solid tumors. Specific targeting of cancer stem cells could provide for a novel strategy to eradicate cancers currently resistant to systemic therapy. ABCB5 is a novel human multidrug resistance mediator recently shown to be expressed by human melanomas and additional malignancies including colon cancer and to be responsible for conferring resistance to chemotherapy in vitro. For example, inhibition of ABCB5 renders normally resistant melanoma cells susceptible to doxorubicin, camptothecin and 5-FU. Subsequent work has shown that ABCB5 identifies cancer stem cells in human malignant melanoma that correlate with clinical disease progression and that can be specifically targeted to abrogate tumor growth. Identification of cancer stem cells with enhanced abundance in more advanced disease but susceptibility to specific targeting via a defining chemoresistance determinant has important implications for cancer therapy. Our most recent studies identify a subset of ABCB5-expressing cells in clinical human colon cancers, established colon cancer cell lines, and show a correlation between ABCB5 expression and clinical colon cancer progression. Based on these findings we hypothesize that the chemoresistance mediator ABCB5, similar to its function in melanoma, identifies colon cancer stem cells and that specific targeting of ABCB5-expressing colon cancer cells may result in abrogation of disseminated disease. Here we propose to (1) Study whether ABCB5 can serve as a novel cancer stem cell marker in colon cancer; (2) Examine the chemoresistance of ABCB5+ colon cancer cells and related cancer stem cell subpopulations to 5-FU and develop strategies for ABCB5-targeted chemoresistance reversal; (3) Investigate whether ABCB5- targeted colon cancer stem cell ablation or chemoresistance reversal can inhibit tumor initiation/progression in vivo. This will be accomplished by studying primary human cancer specimens supported by prognostic and outcome data in murine bioassays in which tumor xenografts develop in a manner that recapitulates naturally occurring disease and in which ABCB5+ tumor cells may be therapeutically targeted. The proposed approaches will determine the clinical relevance and therapeutic importance of the novel biomarker ABCB5 in colon cancer, and should pave the way to successful targeting of ABCB5+ colon CSC in human patients.