Exploring the impact of Klotho on Glycan-Driven Intracellular Pathways and Downstream Signaling in Breast Cancer

Klotho is a protein that has garnered significant attention for its roles as an anti-aging factor and a tumor suppressor. Downregulation of Klotho has been linked to enhanced proliferation and reduced apoptotic in cancer cells across various malignancies. However, overexpression of Klotho exerts inhibitory effects on the colony-forming capacity of breast cancer cells in vitro and reduced aging disorders. Klotho’s enzymatic activity has been a subject of debate. While some studies suggest that klotho acts as an enzyme that cleaves sialic acid from glycolipids and glycoproteins, other studies propose that it functions as a regulatory protein (i.e. through FGF23) without direct enzymatic activity. Furthermore, there is conflicting information regarding Kotho’s sialic acid-containing substrates; while some studies suggest it removes a2-6-linked sialic acids, others argue it can also remove a2-3-linked sialic acids particularly on glycolipids. Research from our lab had demonstrated Klotho’s sialidase activity and its downstream effects on various proteins in the breast cancer cell line MCF-7. Exposure of cells to Klotho with pull-down of proteins using anti-galectin3 antibody, revealed that klotho affected multiple cellular pathways in a time-dependent manner, with differential effects after 5minutes versus 2hours after exposure, as determined by mass spectrometry analysis. We hypothesize that Klotho’s activity is broad affecting multiple proteins from diverse pathways in breast cancer, that eventually lead to the inhibition of cancer growth. To elucidate the mechanisms underlying Klotho`s actions, we focused on its interactions with cell surface glycans and downstream signaling effects in breast cancer. We investigated MCF-7 and MDA-MB-231 cell lines by FACS and Western blot, to demonstrate that Klotho acts as a sialidase that cleaves terminal a2-3/6-linked sialic acids, thereby effecting multiple proteins in these breast cancer cells. Further studies will examine the downstream effects on key proteins, as Syndecan1, CD44, ANXA2, GPC1, Raftlin, S100A10, plasminogen. These studies will allow to decipher the interplay between Klotho, key signaling pathways, and cancer growth. Insights into Klotho`s regulatory role in glycan-mediated signaling pathways may pave the way for innovative glycan-targeted interventions aimed at combating cancer progression and potentially extending a healthy lifespan.