Side-Population and Hedgehog Signaling Analyses on Floating vs Attachment Subpopulations of the CEA-Producing UP-LN1 Cell Line as a Study Model---Identification and Eradication of Cancer Stem Cells

We have previously documented the establishment and preliminary characterization of a CEA-producing carcinoma cell line from a lymph node metastatic lesion (poorly differentiated ca) of the neck of a patient with unknown primary [Chang et al., 2005; see Appendix]. This tumor was likely of adenocarcinoma of GI-tract origin, based on its particular phenotype, CK7-/CK20+/CEA+/SCCA- in both the original clinical lymph node tumor specimen and cultured cells. The persistent appearance of adherent (A cells) and floating (F cells) under normal monolayer culture conditions prompted us to investigate differential properties of these two cell types further. Unexpectedly, A cells were found to be highly susceptible to lymphokine-activated killer (LAK)-mediated cytolysis, whereas F cells were essentially resistant to LAK cell killing. Using DNA microarrays, we identified expression of 17 genes in F cells which were upregulated by ≧2-fold than in A cells. We also identified expression of other 38 genes to be upregulated by ≧2-fold in A cells than in F cells. At least 3 cancer stem cell (CSC) markers were identified in the 17 genes upregulated in F cells, suggesting that CSC and their progenitors are more abundant in F subpopulation. With this background information, we hypothesized that “greater numbers of cancer stem-like cells and their progenitors are in F cells than in A cells, and thus it is important to find ways to eradicate these cells for effective cancer therapeutic strategies for this type of malignancy, i.e. adenocarcinomas of the GI tract.” We here therefore propose to conduct studies with the following specific objectives: 1). To elucidate the mechanism(s) underlying the high sensitivity of A cells but not F cells to be to LAK-mediated cytolysis; 2). To sort out and analyze cancer stem cells (CSC), cancer stem-like cells and their immediate progenitors in F cells and A cells using a side-population assay; 3). Isolated side-populations (SPs) in F and A cells will be compared with respect to the expression of CSC genes, including hedgehog (Hh) pathway related genes, including Ihh, Gli and Ptch. Microarray analysis of SP and NSP (nonSP) cells will be performed as needed. 4). To determine if its expression of Hh genes is associated with clinical lymph node metastasis and tumor progression in GI tumor specimens and colorectal tumor cell lines (i.e., to correlate Gli or Ptch immunochemical positivity with patient prognosis). 5). If any of Ihh, Gli and Ptch; genes identified in GI adenocarcinoma cell lines and tumor specimens is associated with cell proliferation, to determine whether any of these genes, for example, Gli, is necessary for maintaining the proliferation potential, after transfection of siRNA targeting Gli mRNA into F, A or P cells of the UP-LN1 cell line. 6). Search for CSC-sensitive drugs, including CDA-2, to determine if they can selectively eradicate most, if not all, SP cells of the UP-LN1 line or other colorectal cell lines. We feel strongly that CSC may have direct translational importance for cancer therapy.

Project ID:PC9709-0450
External Project ID:NSC97-2314-B182-017