Abstract 4169: A novel, short isoform of the +Tip microtubule binding protein CLIP170 confers taxane resistance in gastric cancer

Abstract

Abstract The microtubule (MT) cytoskeleton is one of the most validated therapeutic targets in clinical oncology, as evidenced by the wide clinical activity of taxanes and other MT inhibitors. However, patients with intrinsic drug resistance do not benefit from taxane chemotherapy, such as patients with diffuse gastric cancer (GC). Currently, the molecular mechanisms underlying clinical taxane resistance remain poorly elucidated. Using a panel of GC cell lines with intrinsic sensitivity or resistance to taxanes, we showed lack of drug-target engagement in the resistant lines, despite unimpaired intracellular accumulation of the drug and in the absence of tubulin mutations or altered tubulin isotype expression. Herein, we show for the first time that the resistant cells expressed a novel, short isoform of the MT plus-end binding protein, CLIP170 (CLIP1). CLIP1 binds to the plus-ends of microtubules (+TIPs) and regulates dynein-mediated MT-based trafficking and the tubulin tyrosination cycle. We showed by both mass-spec proteomics and 5’-RACE that the short CLIP1 isoform (CLIP1S) lacks the first 150 amino acids, thus, missing the first Cap-Gly (Cytoskeleton-Associated Protein-Glycine) domain, which is required for proper +TIP localization. Indeed, confocal microscopy experiments showed that CLIP1S was mislocalized to the microtubule lattice in contrast to the canonical comet-like pattern of CLIP1 seen in taxane sensitive cell lines. CLIP1S expression was specifically correlated with taxane resistance (docetaxel, cabazitaxel) as no correlation was observed with other DNA-damaging agents. Since, CLIP1S expression has never been reported before, to establish causation, we stably knocked down CLIP1 and CLIP1S in taxane-sensitive and resistant cells, respectively. CLIP1SKD entirely reversed taxane-resistance (~300 fold) while CLIP1KD had no effect in the taxane-sensitive cell lines. These data suggest a gain-of-function of CLIP1S that leads to taxane resistance. To study the binding kinetics of taxanes to MTs in the presence or absence of CLIP1S we performed live cell imaging of native cytoskeletons using fluorescently-labeled paclitaxel (Flutax). Our data revealed significantly faster dissociation rates of Flutax from MTs in the resistant cells, indicating transient interaction with MTs. Taxane binding to MTs is a two-step process. First taxanes bind to the MT-pore low affinity surface site, which then facilitates access to the high affinity luminal site. Using a small molecule that binds only to MT-pore site, we showed that we showed that taxol binding to the pore is significantly slower in resistant cells expressing CLIP1S, which together with mislocalization of this variant along the MT lattice suggests that it obstructs access to the MT-pore thus restricting entry of taxane into the lumen of microtubules. This finding will have profound implications for taxane resistance as well as microtubule biology broadly. Citation Format: Prashant V. Thakkar, Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Isabel Barasoain, Jose Fernando Diaz, Manish A. Shah, Paraskevi Giannakakou. A novel, short isoform of the +Tip microtubule binding protein CLIP170 confers taxane resistance in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4169. doi:10.1158/1538-7445.AM2017-4169