Alkylation damage leads to transformation in breast epithelial cells
Alkylating agents, one of the major components of cigarette smoke and cancer chemotherapy, are well known DNA damaging agents as well as known or suspected carcinogens. It is well known that, exposure to such genotoxic agents results in DNA breaks which in turn activates ATM and ATR kinases, key proteins in the DNA Damage Response (DDR) pathway which further phosphorylates their downstream targets, Chk2 and Chk1, respectively. In our study, using N-nitroso-N-ethylurea (NEU), a simple monofunctional SN1 type-DNA ethylating agent, we observed activation of DDR kinases following DNA damage induced by NEU in human cancer cell lines in a dose-dependent as well as temporal manner.
Apart from the DNA damaging effects, alkylating agents have been demonstrated to cause mammary tumors in rodent models. Following exposure to NEU, we investigated the ability of NEU to induce transformation using three-dimensional (3D) cultures of non-transformed MCF0A epithelial cells. 3D cultures aid in distinguishing transformed cells from the non-transformed cells by changes in morphology of the acinar structures formed when these cells are grown on extracellular matrix. Such acini resemble the in vivo breast acini structurally and functionally. In our study we have shown the characteristic features of epithelial-mesenchymal (EMT) where loss of polarity, establishment of mesenchymal characteristics and down-regulation of epithelial like characters were observed in MCF10A cells treated with NEU at the single cell stage. These results gave strong implications of transformation occurring in breast epithelial cells on exposure to NEU.
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Exposure of mammary epithelial cells to the methylating agent, MNU (N-methyl nitrosourea) was shown to cause DNA damage that was sufficient to cause disruption of apico-basal polarity in the breast acini grown as three-dimensional cultures as well as induce epithelial-mesenchymal transition (EMT)-like phenotype. Additionally, aberrant Golgi morphology as well as impaired cellular trafficking was observed in methylation damaged breast acini which was through activation of DNA-PK. Interestingly, inhibiting DNA-PK using a small molecule inhibitor was able to partially rescue the Golgi phenotype and polarity defect as well as EMT-like phenotype confirming methylation damage induced activation of DNA-PK as a major mechanism in mediating cellular transformation.
Platelet Activating Factor (PAF) and its role in breast cancer
Apart from the various environmental factors which influence the induction and progression of cancers, bioactive molecules present in the tissue microenvironment as well as tumor milieu can contribute to the occurrence and progression of the disease. Phospholipid mediators such as lysophosphatadic acid, prostaglandins, platelet activating factor and platelet activating factor-like molecules are secreted by cells of the immune system and are present in the microenvironment. Under chronic inflammatory conditions the proportion of these molecules increases in the adjoining tissues. Given the contribution of chronic inflammation in various cancers, the possibility of these molecules behaving as potential factors for cancer initiation and progression has increased multifold.
PAF acting through PAF-R (Platelet activating factor receptor, a G protein-coupled receptor) has been demonstrated to play important roles in various cancers. Apart from the well-known roles of PAF in inflammation, platelet aggregation various groups have demonstrated the contributing role of PAF in cancer progression. Our recent study, demonstrated the ability of PAF to enhance migration in MDA-MB 231 cells (invasive breast cancer cells). Inhibition of JNK pathway reduced motility of PAF-stimulated and un-stimulated cells suggesting the involvement of JNK pathway in both PAF-induced as well as in the inherent motility of breast cancer cells. PI3K pathway, another pathway well known to play a vital role in cell motility, was investigated to understand its role in motility of PAF-stimulated cells. Surprisingly, we observed that inhibition of this pathway though reduced inherent motility of cells, it could only partially rescue increased motility, thus implying the role of PI3K pathway in inherent motility of cells and a possible role in PAF-induced motility. Further studies are being performed to decipher the pathway through which PAF elicits motility in breast cancer cells.
The possibility of PAF inducing transformation in breast epithelial cells has not yet been explored. We observed that in non-transformed breast epithelial cells, PAF induced formation of abnormal acinar structures when cells were grown as 3D cultures under constant stimulation of PAF. Apart from the increase in the number of cells in the acini, indicative of proliferation or evasion of apoptosis, the acinar structures also showed presence of protrusion-like structures indicative of EMT-like phenotype. Taken together, we demonstrated the ability of PAF to induce transformation of non-transformed breast epithelial cells. These results appeal for further investigations to delineate the pathway and identify novel targets to design novel therapeutics.