The overarching goal of the parent grant is to elucidate the mechanism of taxane resistance mediated by a novel variant of the microtubule (MT) plus-end binding protein CLIP-170. The previously unrecognized variant, namely CLIP-170S, displays unique features and biological properties as compared to the parent protein. We identified that CLIP-170S confers taxane resistance by obstructing the MT-pore on the MT polymer surface, which then inhibits taxane access to its high-affinity luminal binding site. Using a computational, systems-biology approach we further identified the receptor tyrosine kinase (RTK) inhibitor, Imatinib, as a drug predicted to reverse taxaneresistance in gastric cancer. Experimental validation showed that Imatinib in combination with docetaxel, reverses entirely taxane resistance by selective depletion of CLIP-170S. Additional RTK inhibitors, such as ponatinib, sunitinib and axotinib, also downregulate CLIP-170S protein, suggesting a broader class-effect. However, the mechanism by which Imatinib and other RTK inhibitors downregulate CLIP-170S protein expression is not known. In this proposal Eiman will investigate a related but novel angle regarding the modulation of CLIP-170S by Imatinib and other RTK inhibitors, via autophagy. The objective of the current proposal is to elucidate the role of autophagy in drug-induced CLIP-170S depletion. Autophagy is a homeostatic, catabolic degradation process whereby cellular proteins and organelles are engulfed by autophagosomes, digested in lysosomes, and recycled to sustain cellular metabolism (1). Autophagy on one hand acts as a tumor suppressor by preventing the accumulation of damaged proteins and organelles, and on the other hand, as a mechanism of tumor cell survival (2). The cell survival autophagy mechanism is frequently implicated in tumor progression (3), especially in gastric cancer which is the disease focus of the parent grant, where autophagy has been reported to promote disease progression from localized to metastatic gastric cancer (4). Using gastric cancer cell lines expressing CLIP-170S, we found that imatinib selectively depletes this variant sensitizing cells to taxane chemotherapy. Imatinib is FDA approved for the treatment of chronic myelogenous leukemia (CML) driven by the Bcr-Abl fusion protein. To the best of our knowledge, there are no reports implicating Imatinib mechanism of action in the regulation of the MT cytoskeleton which is the target of taxane chemotherapy. Therefore, there is no known mechanistic link between taxane resistance and Imatinib mechanism of action. In the parental grant we proposed signaling and computational studies to identify this link, while the current supplement focuses on the role of autophagy, which is a new line of investigation expected to contribute and expand the parent grant. The current proposal is inspired by a recent report showing that Imatinib treatment downregulates Bcr-Abl protein in CML due to sequestration of Bcr-Abl in autophagosomes (5). Interestingly, the autophagosomes utilize the MT-dynein motor protein transport system to fuse with late endosomes and form an autolysosome (6, 7). The connection between the cytoskeleton and autophagy together with our finding that CLIP-170S is localized on the outer MT surface, and is downregulated by Imatinib led us to hypothesize that Imatinib depletes CLIP-170S protein by inducing its sequestration in autophagosomes. To test this new hypothesis, we propose to:
Aim1 : Determine the mechanistic link between Imatinib-induced autophagy and CLIP-170S selective depletion.
Aim 2. Identify the autophagy regulators that mediate the Imatinib-induced CLIP-170S protein downregulation. Methods and techniques: The mammalian autophagy protein, microtubule-associated protein light chain 3 (LC3), is a known marker of autophagosomes. To determine if Imatinib treatment induces autophagosome formation in the gastric cancer cell lines in our panel we will immunostain endogenous LC3 protein and visualize its distribution and subcellular localization by confocal microscopy. A diffuse cytoplasmic pattern, would be indicative of absence of autophagy while a punctate pattern would primarily represent autophagosomes (8).
