Establishment of Highly Tumorigenic Human Colorectal Cancer Cell Line (CR4) with Properties of Putative Cancer Stem Cells

BACKGROUND: Colorectal cancer (CRC) has the third highest mortality rates among the US population. According to the most recent concept of carcinogenesis, human tumors are organized hierarchically, and the top of it is occupied by malignant stem cells (cancer stem cells, CSCs, or cancer-initiating cells, CICs), which possess unlimited self-renewal and tumor-initiating capacities and high resistance to conventional therapies. To reflect the complexity and diversity of human tumors and to provide clinically and physiologically relevant cancer models, large banks of characterized patient-derived low-passage cell lines, and especially CIC-enriched cell lines, are urgently needed. PRINCIPAL FINDINGS: Here we report the establishment of a novel CIC-enriched, highly tumorigenic and clonogenic colon cancer cell line, CR4, derived from liver metastasis. This stable cell line was established by combining 3D culturing and 2D culturing in stem cell media, subcloning of cells with particular morphology, co-culture with carcinoma associated fibroblasts (CAFs) and serial transplantation to NOD/SCID mice. Using RNA-Seq complete transcriptome profiling of the tumorigenic fraction of the CR4 cells in comparison to the bulk tumor cells, we have identified about 360 differentially expressed transcripts, many of which represent stemness, pluripotency and resistance to treatment. Majority of the established CR4 cells express common markers of stemness, including CD133, CD44, CD166, EpCAM, CD24 and Lgr5. Using immunocytochemical, FACS and western blot analyses, we have shown that a significant ratio of the CR4 cells express key markers of pluripotency markers, including Sox-2, Oct3/4 and c-Myc. Constitutive overactivation of ABC transporters and NF-kB and absence of tumor suppressors p53 and p21 may partially explain exceptional drug resistance of the CR4 cells. CONCLUSIONS: The highly tumorigenic and clonogenic CIC-enriched CR4 cell line may provide an important new tool to support the discovery of novel diagnostic and/or prognostic biomarkers as well as the development of more effective therapeutic strategies

Prostate Cancer Stem Cell-Targeted Efficacy of a New-Generation Taxoid, SBT-1214 and Novel Polyenolic Zinc-Binding Curcuminoid, CMC2.24

Background Prostate cancer is the second leading cause of cancer death among men. Multiple evidence suggests that a population of tumor-initiating, or cancer stem cells (CSCs) is responsible for cancer development and exceptional drug resistance, representing a highly important therapeutic target. The present study evaluated CSC-specific alterations induced by new-generation taxoid SBT-1214 and a novel polyenolic zinc-binding curcuminoid, CMC2.24, in prostate CSCs. Principal Findings The CD133high/CD44high phenotype was isolated from spontaneously immortalized patient-derived PPT2 cells and highly metastatic PC3MM2 cells. Weekly treatment of the NOD/SCID mice bearing PPT2- and PC3MM3-induced tumors with the SBT-1214 led to dramatic suppression of tumor growth. Four of six PPT2 and 3 of 6 PC3MM2 tumors have shown the absence of viable cells in residual tumors. In vitro, SBT-1214 (100nM-1µM; for 72 hr) induced about 60% cell death in CD133high/CD44+/high cells cultured on collagen I in stem cell medium (in contrast, the same doses of paclitaxel increased proliferation of these cells). The cytotoxic effects were increased when SBT-1214 was combined with the CMC2.24. A stem cell-specific PCR array assay revealed that this drug combination mediated massive inhibition of multiple constitutively up-regulated stem cell-related genes, including key pluripotency transcription factors. Importantly, this drug combination induced expression of p21 and p53, which were absent in CD133high/CD44high cells. Viable cells that survived this treatment regimen were no longer able to induce secondary spheroids, exhibited significant morphological abnormalities and died in 2-5 days. Conclusions We report here that the SBT-1214 alone, or in combination with CMC2.24, possesses significant activity against prostate CD133high/CD44+/high tumor-initiating cells. This drug combination efficiently inhibits expression of the majority of stem cell-related genes and pluripotency transcription factors. In addition, it induces a previously absent expression of p21 and p53 (“gene wake-up”), which can potentially reverse drug resistance by increasing sensitivity to anti-cancer drugs

HMGA1 Induces Intestinal Polyposis in Transgenic Mice and Drives Tumor Progression and Stem Cell Properties in Colon Cancer Cells

Although metastatic colon cancer is a leading cause of cancer death worldwide, the molecular mechanisms that enable colon cancer cells to metastasize remain unclear. Emerging evidence suggests that metastatic cells develop by usurping transcriptional networks from embryonic stem (ES) cells to facilitate an epithelial-mesenchymal transition (EMT), invasion, and metastatic progression. Previous studies identified HMGA1 as a key transcription factor enriched in ES cells, colon cancer, and other aggressive tumors, although its role in these settings is poorly understood.To determine how HMGA1 functions in metastatic colon cancer, we manipulated HMGA1 expression in transgenic mice and colon cancer cells. We discovered that HMGA1 drives proliferative changes, aberrant crypt formation, and intestinal polyposis in transgenic mice. In colon cancer cell lines from poorly differentiated, metastatic tumors, knock-down of HMGA1 blocks anchorage-independent cell growth, migration, invasion, xenograft tumorigenesis and three-dimensional colonosphere formation. Inhibiting HMGA1 expression blocks tumorigenesis at limiting dilutions, consistent with depletion of tumor-initiator cells in the knock-down cells. Knock-down of HMGA1 also inhibits metastatic progression to the liver in vivo. In metastatic colon cancer cells, HMGA1 induces expression of Twist1, a gene involved in embryogenesis, EMT, and tumor progression, while HMGA1 represses E-cadherin, a gene that is down-regulated during EMT and metastatic progression. In addition, HMGA1 is among the most enriched genes in colon cancer compared to normal mucosa.Our findings demonstrate for the first time that HMGA1 drives proliferative changes and polyp formation in the intestines of transgenic mice and induces metastatic progression and stem-like properties in colon cancer cells. These findings indicate that HMGA1 is a key regulator, both in metastatic progression and in the maintenance of a stem-like state. Our results also suggest that HMGA1 or downstream pathways could be rational therapeutic targets in metastatic, poorly differentiated colon cancer