Molecular imaging of the kinetics of hyperactivated ERK1/2-mediated autophagy during acquirement of chemoresistance

Aniketh Bishnu,Pratham Phadte,Asmita Sakpal,Ajit Dhadve,Pritha Ray,Bharat Rekhi

doi:10.1038/s41419-021-03451-y

Abstract

Alterations in key kinases and signaling pathways can fine-tune autophagic flux to promote the development of chemoresistance. Despite empirical evidences of strong association between enhanced autophagic flux with acquired chemoresistance, it is still not understood whether an ongoing autophagic flux is required for both initiation, as well as maintenance of chemoresistance, or is sufficient for one of the either steps. Utilizing indigenously developed cisplatin–paclitaxel-resistant models of ovarian cancer cells, we report an intriguing oscillation in chemotherapy-induced autophagic flux across stages of resistance, which was found to be specifically elevated at the early stages or onset of chemoresistance. Conversely, the sensitive cells and cells at late stages of resistance showed stalled and reduced autophagic flux. This increased flux at early stages of resistance was found to be dictated by a hyperactive ERK1/2 signaling, which when inhibited either pharmacologically (U0126/Trametinib) or genetically, reduced p62 degradation, number of LC3+veLAMP1+ve puncta, autophagolysosome formation, and led to chemo-sensitization and apoptosis. Inhibition of ERK1/2 activation also altered the level of UVRAG and Rab7, the two key proteins involved in autophagosome–lysosome fusion. Noninvasive imaging of autophagic flux using a novel autophagy sensor (mtFL-p62 fusion reporter) showed that combinatorial treatment of platinum–taxol along with Trametinib/chloroquine blocked autophagic flux in live cells and tumor xenografts. Interestingly, Trametinib was found to be equally effective in blocking autophagic flux as chloroquine both in live cells and tumor xenografts. Combinatorial treatment of Trametinib and platinum–taxol significantly reduced tumor growth. This is probably the first report of real-time monitoring of chemotherapy-induced autophagy kinetics through noninvasive bioluminescence imaging in preclinical mouse model. Altogether our data suggest that an activated ERK1/2 supports proper completion of autophagic flux at the onset of chemoresistance to endure initial chemotherapeutic insult and foster the development of a highly chemoresistant phenotype, where autophagy becomes dispensable.

Highlights

IntroductionChemoresistance, a multistep dynamic process, involves activation of several key kinase pathways like AMPK, mTOR1, AKT, and ERK1/2, which in turn can modulate and fine-tune the autophagic flux in cancer cells[3,4,5]
Macroautophagy, referred as autophagy, a stressinduced cellular catabolic process is reported to promote acquirement of chemoresistance in various malignancies, including epithelial ovarian cancer (EOC)[1,2].Chemoresistance, a multistep dynamic process, involves activation of several key kinase pathways like AMPK, mTOR1, AKT, and ERK1/2, which in turn can modulate and fine-tune the autophagic flux in cancer cells[3,4,5]
Utilizing a novel autophagy sensor comprised of a mutant thermostable firefly luciferase fused with p62, we further showed that combinatorial treatment of cisplatin-paclitaxel (CisPac) with either Trametinib, a clinically approved MEK1/2 inhibitor or chloroquine (CQ) led to p62 accumulation, while only chemotherapeutic drug treatment led to p62 degradation as monitored longitudinally by bioluminescence imaging in live cells and tumor-bearing mice

Summary

Introduction

Chemoresistance, a multistep dynamic process, involves activation of several key kinase pathways like AMPK, mTOR1, AKT, and ERK1/2, which in turn can modulate and fine-tune the autophagic flux in cancer cells[3,4,5]. In contrast to AKT, which promotes development of chemoresistance in several cancers but negatively regulates autophagy, the MAPK/ERK1/2 signaling plays a contextual role in modulating autophagic flux[6,7,8,9]. ERK1/2 promotes autophagic flux in chemoresistant gastric, breast, and ovarian cancer cells, but downregulates the same in basal or BRAF inhibitor resistant lung, breast, and Official journal of the Cell Death Differentiation Association. PDAC cells[10,11,12] All these studies reported a onetime relation between chemoresistance and autophagic flux, and did not investigate the dynamic association of autophagy across different stages of resistance. Understanding the role of autophagy during the development of chemoresistance and its regulation through associated kinase/s would enable us to design and implement optimal therapeutic regimen at precise therapeutic window to combat chemoresistance

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