ER-Localized Deadenylase PNLDC1 Suppresses Colorectal Cancer Progression by Targeting the mRNA decay of TUBB4B
Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, highlighting the urgent need for novel therapeutic strategies. In this study, we find that PNLDC1, an endoplasmic reticulum (ER)-localized deadenylase, lower expresses in 91 clinical CRC tissues. Using both in vitro and in vivo models, we identify PNLDC1 as a key tumor suppressor by modulating cell cycle progression in CRC. RNA-immunoprecipitation and sequencing analyses reveal that PNLDC1 binds cell cycle-associated mRNAs, with TUBB4B emerging as a critical target. PNLDC1 downregulates TUBB4B mRNA, whose regulated degradation is essential for PNLDC1's tumor-suppressive function. Restoration of TUBB4B expression counteracts PNLDC1-mediated suppression of CRC cell proliferation, underscoring its pivotal role in CRC pathogenesis. Importantly, we demonstrate that the TUBB4B inhibitor, mebendazole, effectively suppresses the enhanced proliferation in lower-expression PNLDC1 models, including cell, mouse xenografts, and patient-derived tumor-like cell clusters, positioning it as a promising therapeutic agent. These findings establish PNLDC1 as a valuable biomarker and therapeutic target, paving the way for developing novel CRC treatments. Overall design: RIP assays were performed following established protocols54. 10 million cells were collected and lysed in RIP buffer (20 mM Tris-HCl pH=7.4, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1% NP-40, 100U RNase inhibitor, 1×cocktail), and lysates were incubated with 6 µg anti-PNLDC1 antibody-coated beads (Proteintech, 25559-1-AP) overnight at 4°C. Immunocomplexes were washed with washing buffer for 3 times (20 mM Tris-HCl pH=7.4, 100 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA), treated with proteinase K, and RNA was extracted using phenol/chloroform/isoamyl alcohol (125:24:1). Clean reads were aligned to the hg38 genome using bowtie2 (v2.5.1). Both input and RIP samples were prepared for next-generation sequencing.