Abstract
5-Fluorouracil (5-FU) remains the gold standard of first-line treatment for colorectal cancer (CRC). Although it may initially debulk the tumor mass, relapses frequently occur, indicating the existence of cancer cells that are therapy-resistant and are capable of refueling tumor growth. To identify mechanisms of drug resistance, CRC stem-like cells were subjected to long-term 5-FU selection using either intermittent treatment regimen with the IC50 drug dose or continuous treatment regimen with escalating drug doses. Parental cancer cells were cultivated in parallel. Real-time PCR arrays and bioinformatic tools were used to investigate gene expression changes. We found the first method selected for cancer cells with more aggressive features. We therefore transplanted these cancer cells or parental cells in mice, and again, found that not only did the 5-FU-selected cancer cells generate more aggressive tumors with respect to their parental counterpart, but they also showed a different gene expression pattern as compared to what we had observed in vitro, with ID1 the top upregulated gene. We propose ID1 as a stemness marker pervasively expressed in secondary lesions emerging after completion of chemotherapy.
Highlights
Current first-line chemotherapy generally consists of cytotoxic agents, like 5-fluorouracil (5-FU), a uracil analogue that following intracellular conversion into three active metabolites causes alterations in RNA processing/function and severe DNA damage [1]
Antiapoptotic mechanisms together with cytoprotective autophagy [4], upregulated DNA repair pathways [5], and metabolic rewiring [6] allow tumor cells to thrive in conditions that otherwise would be lethal propagating the disease
While the cancer stem cell (CSC) theory could explain the hierarchical heterogeneity observed in most tumors, and why some patients suffer relapses after initial successful therapy [11], clonal evolution driven by genomic instability is likely to play an important role in tumor progression by allowing CSCs to adapt to environmental, immunologic and pharmacologic cues [12]
Summary
Current first-line chemotherapy generally consists of cytotoxic agents, like 5-fluorouracil (5-FU), a uracil analogue that following intracellular conversion into three active metabolites (fluorouridine triphosphate, fluorodeoxyuridine triphosphate, and fluorodeoxyuridine monophosphate) causes alterations in RNA processing/function and severe DNA damage [1]. The longstanding cancer model, known as ‘clonal evolution’ postulates that tumors arise from a single mutated cell This cell, in turn, generates an abnormal offspring that mutates, forming a mass of genetically diverse tumor cells [7]. To our knowledge, re-transplantation into an in vivo setting of such in vitro established resistant cell lines has been poorly addressed In this manuscript, we used stem cells that were identified, isolated, and characterized in our laboratory from colorectal cancer (CRC) patients’ specimens [13]. While 5-FU resistant cells under continuous treatment with escalating drug doses were predicted to be more prone to cell death with respect to parental cells, cell cycle progression was not predicted to be delayed in the other cell group despite DNA damage, suggesting a propensity to genomic instability, as previously described [13]. We identified ID1 as the top upregulated gene in the tumor generated by 5-FU resistant cells
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