Modulation of Chemotherapy Resistance through Hypoxia Inducible Factor-1 (HIF-1) Inhibition

Abstract

Cancer is one the leading reason of mortality worldwide. Resistance to chemotherapy i.e. ‘chemotherapy resistance’ shares the bulk of cancer related mortality. This resistance is mediated by many cellular pathway alterations, ensuing cellular hypoxia being one of the well-known factors. The hypoxic mechanism is driven by various genetic signatures, the most notable among which is hypoxia inducible factor-1α (HIF-1α) which regulates transcription of many genes to promote cancer cell survival, and progression. Multidrug resistance gene-1 (MDR1) and Lysosome-associated protein transmembrane 4B-35 (LAPTM4B-35) are among those notable players which augment their responses to cellular hypoxia. Therefore, pharmacological inhibition of HIF-1 by disrupting its dimerization can be a key strategy to overcome therapy resistance primarily and arresting tumor growth and progression secondarily. This thesis dissertation suggests potential HIF1 dimerization inhibitors constructed through ligand- and structure-based pharmacophore modelling with rigorous virtual database screening. The shortlisted hits then underwent cell line testing under hypoxic conditions for validation of HIF1 inhibition and inhibition of down-stream HIF1 effector gene VEGF and GLUT1. The current work also studied co-expression of hypoxia driven genes HIF-1α, MDR1 and LAPTM4B in peripheral blood lymphocytes of chemotherapy receiving 72 breast, 42 ovarian, 32 colon and 21 prostate cancer patients with reference to their correlation of clinic-pathologic parameters. The current work also proposed structural determinants of LAPTM4B gene computationally for its potential functionality and interaction with cellular proteins of PI3-AKT pathway involved in mediating chemotherapy resistance. The results from the computational and in-vitro validation by western blot analysis identified compound 2 and compound 6 to be effectively disrupting dimerization with concentration of 18.4±14.5 and 274±53.5 μM respectively. The co-expression of HIF-1α, MDR1 and LAPTM4B has been statistically scrutinized via Fisher’s Exact test and the Spearman correlation method. The expression analysis suggested 12–13 folds’ increase in expression of HIF-1α, 2-fold increase in MDR1 and 13–14 fold increase in LAPTM4B mRNA level in peripheral blood of breast, ovarian, prostate and colon cancer patients. In the current study there was an association of HIF-1α, MDR1 and LAPTM4B expression Abstract 2 with advanced tumor stage, metastasis and chemotherapy treated group in breast, ovarian, prostate and colon cancer patients. The Spearman analysis also revealed a positive linear association among HIF-1α, MDR1 and LAPTM4B in all the studied cancer patients. The results from LAPTM4B structural characterization and interaction revealed LAPTM4B interaction with P85α (regulatory domain of PI3K) through its PPRP motif while it interacts with NEDD4 through its PY domain. The important positional interactions are Arg26:LAPTM4B and Glu52:P85α, Arg90:LAPTM4B and Asp21:P85α, Leu348:LAPTM4B and Gly421:NEDD4, Glu362, Tyr351:LAPTM4B and Arg430:NEDD4. The current thesis work proposed potential HIF-1 inhibitors which can be structurally optimized for efficacy, selectivity, pharmacokinetic and toxicity profiling before clinical investigations. The elevated expression of HIF-1α, MDR1 and LAPTM4B in peripheral blood of solid tumor patients can be a predictor of metastasis, disease progression and treatment response in cancers. These interactions of LAPTM4B can aid in drug targeting to design novel LAPTM4B inhibitors.