Abstract 3600: Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation

Abstract

Abstract The taxanes (i.e., paclitaxel, docetaxel (DTX) and cabazitaxel) are microtubule (MT)-stabilizing drugs widely used to treat solid tumor malignancies. Their success is limited by the presence of intrinsic or acquired drug resistance. Understanding the molecular mechanisms of taxane resistance is a key to significantly improve clinical outcomes of taxane-based chemotherapy. In gastric cancer (GC), retrospective analysis of the TAX-325 trial revealed that the addition of docetaxel (DTX) to standard cisplatin/fluorouracil increased progression-free and overall survival, primarily only in intestinal subtype (INT) GC, suggesting that diffuse (DIF) GC may be intrinsically taxane resistant. In fact, our previous data supported this hypothesis; DIF GC cell lines had higher incidence (63%) of DTX resistance than INT GC cell lines (25%). Flow cytometric analyses showed negligible P-glycoprotein expression on the cell surface of all cell types, and accumulation of C14-labeled DTX in cells was observed in both sensitive and resistant GC cell lines. These results ruled out drug efflux as a possible mechanism that confers taxane resistance in DIF GC cells. Next, we used fluorescein-conjugated paclitaxel (Flutax) as a probe to investigate the affinity of taxanes to MTs. Live cell imaging showed strong binding of Flutax to MTs in a sensitive cell line at least for 60 minutes. In contrast, Flutax failed to stay bound to MTs in resistant GC cell lines,. In addition, discontinuous decoration of MTs with Flutax was seen in resistant but not in sensitive cell lines. Our observation suggests that MTs of resistant GC cell lines have lower affinity for taxanes, and there might be subtle structural changes of MTs. We are currently conducting the competition of Flutax/DTX to quantify the affinity of DTX in both types of GC cell lines. Because tubulin mutations have been reported in many taxane-resistant cell lines, we sequenced the different tubulin isotypes in GC cell lines using both Sanger and next generation sequencing. No mutations were identified. In addition, we did not see any significant changes in the expression of βIII-tubulin or tubulin post-translational modifications between the sensitive and resistant cell lines. Pathway analyses on the RNA-Seq datasets derived from the panels of sensitive and resistant cell lines before and after taxane treatment is ongoing to identify the key molecular events underlying taxane resistance.Taken together, our data indicate that new molecular pathway(s) or change of overall MT structure and/or function may contribute to the impaired taxane binding to MTs in resistant GC cells. Our study will lead us to novel mechanistic insights in intrinsic drug resistance, and will ultimately allow us to target key molecule(s) to overcome chemo-resistance, the key determinant to improve overall survival of patients. Citation Format: Katsuhiro Kita, Giuseppe Galletti, Kyle Cleveland, Prashant V. Thakkar, Ada Gjyrezi, Chao Zhang, Isabel Barasoain, J. Fernando Díaz, Doron Betel, Manish A. Shah, Paraskevi Giannakakou. Impaired taxane binding to MTs in intrinsically taxane resistant gastric cancer cells without β-tubulin mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3600. doi:10.1158/1538-7445.AM2015-3600