Claudin-4-adhesion signaling drives breast cancer metabolism and progression via liver X receptor β

Yuko Murakami-Nishimagi, Kotaro Sugimoto, Makoto Kobayashi, Kazunoshin Tachibana, Manabu Kojima, Maiko Okano, Yuko Hashimoto, Shigehira Saji, Tohru Ohtake, Hideki Chiba

Cell adhesion is indispensable for appropriate tissue architecture and function in multicellular organisms. Besides maintaining tissue integrity, cell adhesion molecules, including tight-junction proteins claudins (CLDNs), exhibit the signaling abilities to control a variety of physiological and pathological processes. However, it is still fragmentary how cell adhesion signaling accesses the nucleus and regulates gene expression. By generating a number of knockout and rescued human breast cell lines and comparing their phenotypes, we determined whether and how CLDN4 affected breast cancer progression in vitro and in vivo. We also identified by RNA sequencing downstream genes whose expression was altered by CLDN4-adhesion signaling. Additionally, we analyzed by RT-qPCR the CLDN4-regulating genes by using a series of knockout and add-back cell lines. Moreover, by immunohistochemistry and semi-quantification, we verified the clinicopathological significance of CLDN4 and the nuclear receptor LXRβ (liver X receptor β) expression in breast cancer tissues from 187 patients. We uncovered that the CLDN4-adhesion signaling accelerated breast cancer metabolism and progression via LXRβ. The second extracellular domain and the carboxy-terminal Y197 of CLDN4 were required to activate Src-family kinases (SFKs) and the downstream AKT in breast cancer cells to promote their proliferation. Knockout and rescue experiments revealed that the CLDN4 signaling targets the AKT phosphorylation site S432 in LXRβ, leading to enhanced cell proliferation, migration, and tumor growth, as well as cholesterol homeostasis and fatty acid metabolism, in breast cancer cells. In addition, RT-qPCR analysis showed the CLDN4-regulated genes are classified into at least six groups according to distinct LXRβ- and LXRβS432-dependence. Furthermore, among triple-negative breast cancer subjects, the "CLDN4-high/LXRβ-high" and "CLDN4-low and/or LXRβ-low" groups appeared to exhibit poor outcomes and relatively favorable prognoses, respectively. The identification of this machinery highlights a link between cell adhesion and transcription factor signalings to promote metabolic and progressive processes of malignant tumors and possibly to coordinate diverse physiological and pathological events.