Adhesion-dependent and Mechanosensitive regulation of Golgi organization and function in breast cancer cells

Abstract

Cell-matrix adhesion is fundamental to cell survival, signalling, and mechanosensing, and its dysregulation is a hallmark of cancer progression. In breast cancer, progressive extracellular matrix stiffening profoundly remodels cytoskeletal architecture; however, how mechanical cues are transmitted to intracellular organelles to regulate their organization and function remains poorly understood. This thesis addresses this gap by defining an adhesion-dependent pathway that links matrix stiffness sensing to Golgi organization and function in breast cancer cells. Using breast cancer cell lines with distinct invasive properties, this work demonstrates that Golgi organization is highly dynamic and governed by both cell-matrix adhesion and matrix stiffness. In highly invasive MDAMB231 cells, the Golgi is organized and perinuclear under adherent conditions but rapidly disorganizes upon adhesion loss. Increasing matrix stiffness progressively enhances Golgi organization in these cells, accompanied by increased tubulin acetylation. In contrast, non-invasive MCF7 cells display a constitutively disorganized Golgi that is largely insensitive to changes in adhesion or stiffness, highlighting intrinsic differences in Golgi mechano-regulation between these breast cancer subtypes. An integrative in silico analysis of differentially expressed Golgi-associated genes between MDAMB231 and MCF7 cells- combining CCLE transcriptomic data, STRING protein interaction networks, and literature curation- identified the receptor tyrosine kinase AXL as a putative regulator of differential of Golgi organization. AXL is differentially expressed between the two cell lines and prominently localizes to the Golgi in MDAMB231 cells. Functional perturbation of AXL through pharmacological inhibition or knockdown, abrogates stiffness-dependent Golgi organization in MDAMB231 cells. Conversely, expression of AXL in MCF7 cells restores Golgi organization specifically at higher-stiffness. Mechanistically, this study identifies AXL as a vital regulator of the small GTPase Arf1, whose activation and Golgi localization are essential for Golgi integrity and function. Matrix stiffness causes coordinated increases in AXL and Arf1 levels, leading to enhanced Arf1 activation and Golgi localization. Loss of adhesion disrupts this axis, resulting in reduced Arf1 activity, displacement of both AXL and active Arf1 from the Golgi, and subsequent Golgi disorganization. Disruption of this pathway has functional consequences, including reduced tubulin acetylation and altered cell-surface glycosylation, underscoring the broader impact of adhesion-dependent Golgi regulation on cell functions. Collectively, this work defines a mechanoresponsive adhesion-AXL-Arf1 signalling axis that integrates extracellular matrix cues with Golgi organization and function in breast cancer cells. These findings uncover an underappreciated role for mechano-regulation of the Golgi and establish AXL as a critical molecular link between adhesion signalling, Golgi organization, and cancer cell mechanobiology. Keywords: Cell-matrix adhesion, Golgi organization, Matrix stiffness, AXL-Arf1 signalling axis, Mechanosensing