Mechanisms Of Resistance To Antitumor Agents Targeting PI3K

Background:Drug resistance is a critical challenge attenuating the therapeutic efficacy ofmolecule targeted anti-cancer drugs, which is also one of causes leading to failure inclinical trials. Research on the mechanisms underlying the resistance to moleculetargeted agents becomes an important field in tumor biology and therapeutics.Phosphatidylinositol3-kinase (PI3K) family members play an important role intumorigenesis by participating many vital biological processes including cell growth,survival, proliferation, angiogenesis, migration and metastasis. The PI3K pathwayis frequently deregulated in numerous tumor types due to genetic and epigeneticaberrations, leading to its hyperactivation. Thus, a number of candidate drugs areunder active clinical trials agaist multiple types of cancer. PI3K is in the hub of ahuge cell signaling transduction networks and crosstalks with multiple importantsignaling pathways. Tumor cells likely to acquire resistance to PI3K inhibitors aftercontinuous drug exposure. Characterize the mechanisms of resistance to anticancerdrugs targeting PI3K is of great significance, providing not only strategies toovercome the clinical tolerance that may occur, but also useful clues for rational useof these inhibitors.Objectives:This project aims to establish human breast cancer cells (SKBR3) line resistantto PI3K-selective inhibitors and explore the potential molecular mechanisms of theacquired resistance. We sought to identify therapeutic strategy based on the resistancemechanism and provide theoretical and experimental basis for the rational use ofPI3K inhibitors in clinical settings.Methods:CYH33, a PI3K isoforms-selective inhibitor discovered in our laboratory, wasused as the tool compound to establish drug-resistant cell line SKBR3/CYH33bystepwise increasing concentrations of CYH33. Morphological changes of the cellswere observed with microscope. SRB assay was used to detect the sensitivity of parental and resistant cells to various anti-cancer agents and then the resistance indexwas calculated. Cell proliferation curves were plotted by counting cell number toobtain cell doubling time. The cell size and cell cycle distribution were detected byFlow cytometry (FCM). The amount of CYH33in cells was analyzed with HPLC.Microarray technology was employed to compare the mRNA level of the wholetranscriptome of the parental and CYH33-resistant cells. Differentially expressedgenes that closely related to the PI3K/mTOR signaling pathway were furthervalidated using real-time quantitative PCR. Western blot was used to detect thechanges of main molecules downstream of PI3K pathway in parental cells andresistant cells after CYH33treatment, dissecting possible alteration in signalingpathways mediating drug resistance. Combination of CYH33with different drugs wastested to improve the sensitivity of resistant cells to CYH33. All data aredemonstrated as mean±standard deviation (Mean±SD). Microsoft Excel andGraphPad Prism were used for data processing. P value of two groups was calculatedby student’s t-test, where P>0.05, P<0.05are designated as no statistical significanceand statistical significance respectively.Results:SKBR3/CYH33cells were established after4-months-induction, which growand proliferate stably in the medium containing10μM CYH33. These cells exhibiteda45-fold resistance to CYH33compared to parental SKBR3cells and becamerounder and larger. Cell population in S phase remained unchanged in SKBR3andSKBR3/CYH33, but the proportion in G2/M phase increased significantly alongwith decreased population in G1/G0phase. However, no difference in proliferativerate was observed in both types of cells. SKBR3/CYH33cells showed significantresistance to PI3K isoform selective inhibitors (CYH33and A66) and moderateresistance to pan-PI3K inhibitor (GDC0941), while the sensitivity to the dualPI3K/mTOR or mTOR inhibitors (NVP-BEZ235and AZD6244) were comparable toparental cells. The intracellular drug concentration had no significant differencebetween SKBR3/CYH33cells and SKBR3cells. Microarray analysis showed anumber of genes were differentally transcripted between the two cell lines. Wevalidated enhanced expression of chemokine CCL2/MCP-1in CYH33-resistant cells, but neutralization or down-regulation of CCL2failed to sensitize SKBR3/CYH33cellto CYH33.Though CYH33was able to inhibit the phosphorylation of AKT inSKBR3/CYH33cells, CYH33possessed reduced activity to inhibit thephosphorylation of molecules downstream of mTOR such as p70S6K1, rpS6and4EBP1. Combination of rapamycin or AZD8055significantly improved thesensitivity of resistant cells to CYH33.Conclusions:The PI3K-selective inhibitor resistant cell line SKBR3/CYH33wassuccessfully constructed and it could be used to study the mechanism of drugresistance. Though SKBR3/CYH33cells showed significant morphological changesafter long-term CYH33exposure, cell proliferative rate was comparable to that of theparental cells, suggesting that the resistance to CYH33was not due to alteration incells proliferation. SKBR3/CYH33cells were selectively resistant to PI3K-selectiveinhibitors, with less resistance to pan-PI3K inhibitors and no resistance to mTORinhibitors. Resistance of SKBR3/CYH33cells to CYH33was not due to enhanceddrug efflux or increased expression CCL2/MCP-1. The activation of mTOR and itsdownstream effectors in SKBR3/CYH33cells were no longer affected by PI3Kinhibition, suggesting that mTOR activation independent of PI3K played an importantrole in acquired resistance to PI3K-selective inhibitor. Combination of mTORinhibitor was a feasible way to improve the sensitivity of the drug resistant cells to thePI3K inhibitors.