Investigating the mechanisms of cancer drug resistance

Abstract

Clinical chemo-resistance has posed a challenge to scientists and clinicians since the development of chemotherapeutics as anticancer strategy. Mechanisms of drug resistance continue to be a subject investigation amongst scientific community. Cancer cells employ various strategies to evade drug mediated damage. One of the most common strategies is over expression of ATP-binding cassette transporters (ABC). ABC transporters are energy dependent, membrane bound efflux pumps that transport xenobiotic compounds, toxins and drugs out of cells. Cancer cells mainly over express three ABC transporter, namely, ABCB1, ABCC1, and ABCG2. Although several ABC transporter inhibitors have been generated there are no effective anti-ABC transporter therapeutic molecules in clinic, chiefly due to their side effects. Hence, identification of new regulatory pathways that affect ABC transporter transcription can provide unique therapeutic intervention.

To study drug resistance a drug-resistant cell line model using HeLa cervical cancer cells called HeLa Dox2 was developed in the lab. The cells were developed prolonged exposure to doxorubicin, up to a maximum of 2 µM doxorubicin. In this study, we identified vast overexpression of ABCB1 in HeLa Dox2 cells. Inhibition or knockdown of ABCB1 increased drug retention and rendered them drug sensitive, thus establishing ABCB1 overexpression as the major cause for their drug resistance phenotype. We used this model to dissect the molecular pathways that may be involved in ABCB1 up-regulation. We found c-Jun overexpression in HeLa Dox2 cells contributed to ABCB1 over-expression. We also identified regulatory mechanisms affecting c-Jun acetylation and consequent activation. We found that SIRT1 mediated deacetylation of c-Jun enhances its activation, leading to ABCB1 transcriptional increase. We further identified a role for AMPK signalling in c-Jun activation via changes in its acetylation levels. Inhibition of AMPK/SIRT1/c-Jun pathway led to increase in chemo-sensitivity in HeLa Dox2 cells. We also identified that this pathway is specific to drug resistant cell derivative and does not regulate ABCB1 expression in parental HeLa cells. This further enhances the ‘druggability’ of AMPK/SIRT1/c-Jun pathway in drug resistant cells. Since literature provides evidence that drug resistance and stemness are interlinked in cancer cells, we also used the drug resistance cell line model to study stemness properties of drug resistant cells. We found that HeLa Dox2 had higher stem-like properties including expression of Bmi1, a stemness factor. We found a novel pathway wherein ABCB1 modulated Bmi1 levels in drug resistant cells. We speculate that ABCB1 could regulate Bmi1 levels by regulating its transcription, directly or indirectly. In conclusion, we identified novel regulatory pathways that regulate ABCB1 expression and stemness in drug resistant cells. We suggest that these pathways can be used to ‘drug’ a subset of chemo-resistant cancers.