Cooperative pro-tumorigenic adaptation to oncogenic RAS through epithelial-to-mesenchymal plasticity.

In breast cancers, aberrant activation of the RAS/MAPK pathway is strongly associated with mesenchymal features and stemness traits, suggesting an interplay between this mitogenic signaling pathway and epithelial-to-mesenchymal plasticity (EMP). By using inducible models of human mammary epithelial cells, we demonstrate herein that the oncogenic activation of RAS promotes ZEB1-dependent EMP, which is necessary for malignant transformation. Notably, EMP is triggered by the secretion of pro-inflammatory cytokines from neighboring RAS-activated senescent cells, with a prominent role for IL-6 and IL-1α. Our data contrast with the common view of cellular senescence as a tumor-suppressive mechanism and EMP as a process promoting late stages of tumor progression in response to signals from the tumor microenvironment. We highlighted here a pro-tumorigenic cooperation of RAS-activated mammary epithelial cells, which leverages on oncogene-induced senescence and EMP to trigger cellular reprogramming and malignant transformation.

Snail family members unequally trigger EMT and thereby differ in their ability to promote the neoplastic transformation of mammary epithelial cells.

By fostering cell commitment to the epithelial-to-mesenchymal transition (EMT), SNAIL proteins endow cells with motility, thereby favoring the metastatic spread of tumor cells. Whether the phenotypic change additionally facilitates tumor initiation has never been addressed. Here we demonstrate that when a SNAIL protein is ectopically produced in non-transformed mammary epithelial cells, the cells are protected from anoikis and proliferate under low-adherence conditions: a hallmark of cancer cells. The three SNAIL proteins show unequal oncogenic potential, strictly correlating with their ability to promote EMT. SNAIL3 especially behaves as a poor EMT-inducer comforting the concept that the transcription factor functionally diverges from its two related proteins.

Weekly administration of paclitaxel induces long-term aneugenicity in nude mice.

We investigated the potential in vivo aneugenic effects associated with paclitaxel treatment. For this purpose, we treated female nude mice with paclitaxel using doses equivalent to those used in weekly schedules at the clinical level (three cycles of 30 mg/kg/week for three consecutive weeks followed by one resting week). We then evaluated the frequencies of micronucleated erythrocytes (MNE) in peripheral blood using the acridine orange micronucleus assay. The frequency of MNE was evaluated after 24 h and 168 h of administration of the last dose of each paclitaxel cycle (STA mice group) as well as after one year of the first dose of treatment (LTA mice group). We also analyzed the cytology of peripheral blood and bone marrows obtained from these mice at each time period. In the STA mice group, three cycles of paclitaxel induced a 2.4-fold increase in MNE frequencies compared to the control group (p < 0.01). This effect was observed after 24 h of the last dose of each chemotherapy cycle and persisted at least for 168 h. In the LTA mice group, paclitaxel-treated mice presented a 1.8-fold increase in the MNE frequency (p = 0.01) indicating that paclitaxel-induced MNE increase lasted for at least one year. Although the appearance of micronuclei in erythrocytes and granulocytes in peripheral blood and bone marrow cytological smears, there was no evidence of myeloproliferative disease. The present data therefore indicate an aneugenic potential of paclitaxel for humans, which should be considered in the risk-benefit analysis of its increasing clinical use.

A p21/WAF1 mutation favors the appearance of drug resistance to paclitaxel in human noncancerous epithelial mammary cells.

We investigated the mechanisms responsible for paclitaxel resistance in HME-1 cells (human mammary epithelial cells immortalized with hTERT). These cells were exposed to paclitaxel (10 pM for 7 days) and 20 cellular surviving populations (PSP) were obtained. PSP demonstrated high levels of resistance to paclitaxel cytotoxicity as compared with HME-1 cells. Activation of mdr-1 gene expression was observed in 2 PSP. Protein expression analysis using a C-terminal targeted antibody showed that 13 PSP were negative for p21/WAF1 expression after ionizing radiation (6 Gy) or doxorubicin (100 nM) treatment. Sequencing of the 3 exons of the CDKN1A gene revealed that 13 PSP contained a point mutation in exon 2. This mutation consisted in a T insertion at codon 104 leading to a premature STOP codon appearance. Mismatch amplification mutation assay and RFLP-PCR confirmed the presence of the mutation in 16 PSP. Western blot using an N-terminal targeted antibody demonstrated that the C-terminal-truncated p21/WAF1 protein (14 kDa) was indeed expressed in the 13 PSP. Our data suggest that p21/WAF1 inactivation may confer a strong resistance to paclitaxel in noncancerous breast epithelial cells harboring a p21/WAF1 mutant.