Breast cancer stem cell-like cells generated during TGFß-induced EMT are radioresistant.

Failure of conventional antitumor therapy is commonly associated with cancer stem cells (CSCs), which are often defined as inherently resistant to radiation and chemotherapeutic agents. However, controversy about the mechanisms involved in the radiation response remains and the inherent intrinsic radioresistance of CSCs has also been questioned. These discrepancies observed in the literature are strongly associated with the cell models used. In order to clarify these contradictory observations, we studied the radiosensitivity of breast CSCs using purified CD24-/low/CD44+ CSCs and their corresponding CD24+/CD44low non-stem cells. These cells were generated after induction of the epithelial-mesenchymal transition (EMT) by transforming growth factor ß (TGFß) in immortalized human mammary epithelial cells (HMLE). Consequently, these 2 cellular subpopulations have an identical genetic background, their differences being related exclusively to TGFß-induced cell reprogramming. We showed that mesenchymal CD24-/low/CD44+ CSCs are more resistant to radiation compared with CD24+/CD44low parental cells. Cell cycle distribution and free radical scavengers, but not DNA repair efficiency, appeared to be intrinsic determinants of cellular radiosensitivity. Finally, for the first time, we showed that reduced radiation-induced activation of the death receptor pathways (FasL, TRAIL and TNF-α) at the transcriptional level was a key causal event in the radioresistance of CD24-/low/CD44+ cells acquired during EMT.

Forced extinction of CD24 stem-like breast cancer marker alone promotes radiation resistance through the control of oxidative stress.

Along with CD44, CD24 is a key marker of breast cancer stem cells (CSCs), frequently defined by CD24(-)/CD44(+) labeling. Among all phenotypes classically attributed to breast CD24(-)/CD44(+) cancer cells, radiation resistance has been extensively described and seen as being implicated in radiotherapy failure. Our previous data indicated that CD24(-) cells constitute a radiation-resistant subpopulation transitory selected by high doses of ionizing radiation. However, little is known about the biological role of CD24 in breast cancers, and no function has been assigned to CD24 in radiation response. Here, CD24 expression was induced in CD24(-) cells or knocked-down in CD24(+) cells. We show that forced extinction of CD24 expression is associated with decreased proliferation rate, lower levels of reactive oxygen species (ROS) and decreased genomic instability. On the opposite when CD24 is artificially expressed in CD24(-) cells, proliferation rates in vitro and in vivo, ROS levels and genomic instability are enhanced. Moreover, we observe that loss of CD24 expression leads to radiation resistance, by preventing radiation-induced cell death and promoting generation of progeny in relation to lower G2/M blockade and a smaller proportion of polyploid cells. Finally, control of ROS levels appears to be the key event in the CD24-mediated radiation response. For the first time, CD24 is proposed as a direct actor in radiation response of breast cancer cells, independently of CD44 expression. These findings could have interesting applications in evaluating the intrinsic radiation response of primary tumors.

A new phosphorylated form of Ku70 identified in resistant leukemic cells confers fast but unfaithful DNA repair in cancer cell lines.

Ku70-dependent canonical nonhomologous end-joining (c-NHEJ) DNA repair system is fundamental to the genome maintenance and B-cell lineage. c-NHEJ is upregulated and error-prone in incurable forms of chronic lymphocytic leukemia which also displays telomere dysfunction, multiple chromosomal aberrations and the resistance to DNA damage-induced apoptosis. We identify in these cells a novel DNA damage inducible form of phospho-Ku70. In vitro in different cancer cell lines, Ku70 phosphorylation occurs in a heterodimer Ku70/Ku80 complex within minutes of genotoxic stress, necessitating its interaction with DNA damage-induced kinase pS2056-DNA-PKcs and/or pS1981-ATM. The mutagenic effects of phospho-Ku70 are documented by a defective S/G2 checkpoint, accelerated disappearance of γ-H2AX foci and kinetics of DNA repair resulting in an increased level of genotoxic stress-induced chromosomal aberrations. Together, these data unveil an involvement of phospho-Ku70 in fast but inaccurate DNA repair; a new paradigm linked to both the deregulation of c-NHEJ and the resistance of malignant cells.