Dissecting the role of oncogenes and metabolism in a cell survival following extracellular matrix-detachment using a 3D breast model

Abstract

The ability of breast cancer cells to evade cell death following detachment from the extracellular matrix (ECM) is an important feature of breast cancer development and metastasis. ECMdetachment is traditionally viewed to induce the programmed cell death pathway anoikis, however more recently a defect in metabolism has also been implicated.

The normal breast duct consists of a polarised epithelium surrounding a hollowed lumen, and the MCF-10A cell line can be cultured in 3D to form phenotypically similar acini. The hollowed lumen is formed due to luminal cell death following ECM-detachment; these acini therefore provide a more physiologically relevant setting to examine cell death upon detachment than traditional 2D culture. I utilised this system to study the role of Bcl-2 associated anthogene 1 (Bag-1) overexpression in ECM-detached cells. In addition, HER2-overexpressing MCF-10A (MCF10A:HER2), which yield filled acini structures characteristic of a breast cancer precursor, DCIS, were used alongside non-transformed MCF-10A to examine the effect of molecular and pharmacological alteration of metabolism. Some of the work was extended to primary HMECderived acini to enhance understanding.

Bag-1 is an antiapoptotic protein frequently overexpressed in breast cancer and exists as three differentially localised, functionally discrete isoforms (Bag-1S, Bag-1M, Bag-1L). Overexpression of Bag-1L in stable clones led to acini with filled lumens and an external branching structure. The branching morphology correlated with the level of Bag-1L overexpression and treatment with a small-molecule inhibitor of Bag-1, Thio-2, reduced the level of branching observed. When Bag-1S or Bag-1L isoforms were individually overexpressed in retroviral pooled populations of MCF-10A it led to a temporal delay in the onset of luminal clearing however I did not observe a branching morphology.

Glycolysis and fatty acid oxidation are being targeted therapeutically in cancer as these metabolic pathways are upregulated in cancer cells to promote transformation. Metabolic inhibitors targeting glycolysis (oxamate) and fatty acid oxidation (etomoxir) reversed the HER2-driven transformation of acini. Activation of mitochondrial activity (dichloroacetate) had no effect on MCF10A:HER2 acini but drove controls towards a highly transformed phenotype. This phenotype seen in 3D was not observed in 2D culture. In addition to the pharmacological consequences of metabolic inhibition, overexpression of the metabolic sensor CtBP2 interfered with acinar morphogenesis and promoted transformation which was reversible by CtBP inhibition.

In conclusion, the identification of phenotypes in 3D that were not observed in 2D reinforces the importance of 3D culture systems to examine breast tumourigenesis and pharmacological agents. Bag-1 is able to delay the onset of luminal clearing in MCF-10A acini. In models where Bag-1 is able to fully prevent luminal clearing I propose that a second (unidentified) transformation event has occurred. Metabolic manipulation alters cell survival in ECM-detachment in MCF-10A, highlighting the relevance of metabolism in cancer development and progression and has potential for cancer therapeutics.

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Identifiers

ORCID for Caroline Rose Barker: orcid.org/0000-0001-8263-4151

ORCID for Jeremy Blaydes: orcid.org/0000-0001-8525-0209

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