Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Several models have been developed to study cancer, such as rodent models and established cell lines. Valuable insights into carcinogenesis have been ...

Indian Institute of Science Education and Research

New York University

The DNA damage checkpoint signalling cascade sense damaged DNA and coordinates cell cycle arrest, DNA repair, and/or apoptosis. However, it is still not well understood how the signalling system differentiates between different kinds of DNA damage. N-nitroso-N-ethylurea (NEU), a DNA ethylating agent induces both transversions and transition mutations.

N-nitroso-N-ethylurea activates DNA damage surveillance pathways and induces transformation in mammalian cells.

A plethora of studies have demonstrated that chronic inflammatory microenvironment influences the genesis and progression of tumors. Such microenvironments are enriched with various lipid mediators. Platelet activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is one such lipid mediator that is secreted by different immune cell types during inflammation and by breast cancer cells upon stimulation with growth factors. Overexpression of PAF-receptor has also been observed in many other cancers. Here we report the possible roles of PAF in tumor initiation and progression. MCF10A, a non-transformed and non-malignant mammary epithelial cell line, when grown as 3D 'on-top' cultures form spheroids that have a distinct hollow lumen surrounded by a monolayer of epithelial cells. Exposure of these spheroids to PAF resulted in the formation of large deformed acinar structures with disrupted lumen, implying transformation. We then examined the response of transformed cells such as MDA-MB 231 to stimulation with PAF. We observed collective cell migration as well as motility at the single cell level on PAF induction, suggesting its role during metastasis. This increase in collective cell migration is mediated via PI3-kinase and/or JNK pathway and is independent of the MAP-kinase pathway. Taken together this study signifies a novel role of PAF in inducing transformation of non-tumorigenic cells and the vital role in promotion of breast cancer cell migration.

Platelet-activating factor promotes motility in breast cancer cells and disrupts non-transformed breast acinar structures.

DNA alkylating agents form the first line of cancer chemotherapy. They not only kill cells but also behave as potential carcinogens. MNU, a DNA methylating agent, is well known to induce mammary tumours in rodents. However, the mechanism of tumorigenesis is not well understood. Our study reports a novel role played by DNA-dependent protein kinase (DNA-PK) in methylation damage-induced transformation using three-dimensional breast acinar cultures. Here, we report that exposure of breast epithelial cells to MNU inhibited polarisation at the basolateral domain, increased dispersal of the Golgi at the apical domain and induced an epithelial-to-mesenchymal transition (EMT)-like phenotype as well as invasion. This altered Golgi phenotype correlated with impaired intracellular trafficking. Inhibition of DNA-PK resulted in almost complete reversal of the altered Golgi phenotype and partial rescue of the polarity defect and EMT-like phenotype. The results confirm that methylation damage-induced activation of DNA-PK is a major mechanism in mediating cellular transformation.This article has an associated First Person interview with the first author of the paper.

DNA-dependent protein kinase plays a central role in transformation of breast epithelial cells following alkylation damage.

Breast cancer is the most devastating disease among females globally. Conventional chemotherapeutic regimen relies on the use of highly cytotoxic drugs as monotherapy and combination therapy leading to severe side effects to the patients as collateral damage. Moreover, combining hydrophobic and hydrophilic drugs create erratic biodistribution and suboptimal medicinal outcome. Hence, packaging multiple drugs of diverse mechanisms of action and biodistribution for safe delivery into tumor tissues with optimal dosages is indispensable for next-generation breast cancer therapy. To address these, in this report, we describe a unique cisplatin-triggered self-assembly of linear polymer into 3D-spherical sub 200 nm particles. These nanoparticles comprise a hydrophobic (paclitaxel) and hydrophilic drug (cisplatin) simultaneously in a single particle. Molecular dynamics simulation revealed hydrophilic-hydrophilic interaction and interchain H-bonding as underlying mechanisms of self-assembly. Confocal microscopy studies evidently demonstrated that these novel nanoparticles can home into lysosomes in breast cancer cells, fragment subcellular nuclei, and prevent cell division, leading to improved breast cancer cell death compared to free drug combination. Moreover, 3D-breast tumor spheroids were reduced remarkably by the treatment of these nanoparticles within 24 h. These dual-drug-loaded self-assembled polymeric nanoparticles have prospective to be translated into a clinical strategy for breast cancer patients.

Drug-Triggered Self-Assembly of Linear Polymer into Nanoparticles for Simultaneous Delivery of Hydrophobic and Hydrophilic Drugs in Breast Cancer Cells.

Lipid species are known to have various biological functions owing to their structural differences, and each of them possesses a specific role to play depending upon their location and distribution in the cell. Some of these lipids interact with proteins on the cell membrane and acts as second messengers. The level of lipid mediators is generally maintained in the cell by feedback mechanisms; however, their improper degradation or enhanced production leads to their accumulation in the tumor microenvironment and disturbs the homeostasis of the cell. Platelet activating factor (PAF) is a known phospholipid mediator secreted upon immunological challenges by platelets, neutrophils, basophils, and macrophages. PAF, as a potent inflammatory molecule, is well studied, and its role in various cancers and cardiovascular diseases has also been investigated. Interestingly, increased levels of PAF have been found in the blood plasma of smokers, and breast cancer cells have shown the accumulation of PAF in presence of cigarette smoke extract. This accumulation was found to increase tumor cell motility that in turn could promote metastasis. Beyond this, however, the effect of PAF on tumorigenesis has not yet been well explored. Here, we show that the continuous exposure of 3D breast acinar cultures to PAF resulted in the activation of various oncogenic signaling pathways leading to transformation. We also found that the presence of PAF in the micro-environment increased the expression of PAF receptor (PAF-R), which corroborated with the higher expression of PAF-R detected in some epithelial cancers, as per literature. Thus, this study impresses on the fact that the presence of PAF alters the cellular microenvironment and eventually triggers irreversible effects that can cumulatively lead to transformation.

Libi Anandi

Biology Department

Indian Institute of Science Education and Research

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Libi Anandi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Biology Department

Indian Institute of Science Education and Research

Phospholipid Mediator Induced Transformation in Three-Dimensional Cultures

Biology Department