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The role of autophagy in anticancer therapy

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posted on 2024-09-03, 05:27 authored by Matheus Dyczynski

Autophagy is a fundamental catabolic process, which is utilized by nearly every cell and tissue type upon stress exposure and has been shown to contribute to resistance to chemotherapy in a variety of cancers. The subject of this thesis is to shed light on the role of autophagy in chemotherapy and to investigate novel regulators of autophagy.

Multiple clinical trials have been started in order to overcome resistance to standard therapy by combining it with lysosomal inhibitor hydroxychloroquine, yet with limited success. This drug has been shown to have poor cell uptake properties in solid tumors due to tumor acidosis. In paper I we found that the compound Salinomycin is a potent autophagy inhibitor in multiple cancer cell lines, especially under acidic conditions. Salinomycin was able to penetrate the acidic core of multicellular spheroids and decrease cell viability and clonogenic survival of colorectal cancer cells. We also show that Salinomycin efficiently blocked autophagic flux in breast cancer cells. In particular, cancer stem cells derived from cell lines or primary breast cancer tumors showed reduced viability and reduced capability to form mammospheres under Salinomycin treatment. Using mass spectrometry, we could confirm pH-dependent intracellular accumulation of Salinomycin. This data proves the potency of Salinomycin as an anti-cancer drug with capacities to modulate autophagy in the acidic tumor microenvironment.

Part of the standard treatment regimen of pediatric patients with Acute Lymphoblastic Leukemia (ALL) are glucocorticoids (GC). This metabolic hormone is effective in inducing cell death in ALL cells. GC mediated inhibition of glucose uptake and upregulation of catabolic processes such as autophagy have previously been reported. In paper II we addressed in detail what metabolic changes occur upon GC treatment in ALL cell lines by parallel time-course proteomics, metabolomics and isotope tracing, and by confirming selected findings by cross-referencing with publicly available microarray data and experimentally by qRT-PCR. Our findings confirmed the onset of growth arrest, autophagy and apoptosis. Not only glucose but also glutamine entry into the Citric-Acid-Cycle was inhibited contrasting the upregulation of glutamine-ammonia-ligase (GLUL) expression suggesting the induction of glutamine synthesis. Potentiating the GLUL-mediated reaction rescued cell viability and reduced autophagic flux suggesting that GLUL induction and glutamine synthesis are relevant for the autophagy induction and sensitivity of ALL cells to GCs. This data provides a comprehensive overview of metabolic changes in ALL cells upon GCs' treatment and may shed light on the mechanism of GC-induced cell death in ALL cells.

In paper III we used high-content microscopy to screen the FIMM drug library consisting of 306 anticancer drugs and identified 104 autophagy modulators, of which 16 showed cell death potentiation upon siRNA mediated knock-down of ATG7 (autophagy-related protein 7) and VPS34 (vacuolar protein sorting 34), key regulators of autophagy. We validated the hits in 2 breast cancer cell lines, MDA-MB231 and MCF7, and continued to characterize two of the hits, Erlotinib and Sunitinib, in detail. The collaboration with Sprint Bioscience led to the development of SB02024, a specific inhibitor of the VPS34 kinase. We showed that SB02024 could block autophagy in vitro and in in vivo xenograft mouse models. Combination of SB02024 with Sunitinib and Erlotinib increased cytotoxicity by these drugs in either 2D cell culture, colony formation assays, or, in case of Sunitinib, in cells grown in 3D as multicellular spheroids. This data further strengthens the notion that using VPS34 inhibitors in combination with targeted tyrosine kinase inhibitor-based therapy, and particularly Sunitinib, can overcome resistance and emphasizes their value in cancer treatment.

RAS protein activator like 2 (RASAL2) is a known tumor-suppressor regulating members of the RAS-family of oncoproteins. In paper IV we describe for the first time a role for RASAL2 in the induction of autophagy. We found that autophagy induction via pharmaceutical mTOR inhibition or amino acid-starvation increased RASAL2 transcription. Furthermore, RASAL2 protein levels were regulated by autophagy-dependent protein degradation. Thus, in the starved cells, RASAL2 mRNA levels were induced while protein levels declined. Also, depletion of autophagy-related protein 7 (ATG7) that impaired autophagy process resulted in a striking increase in RASAL2 protein levels. RNAi-mediated knockdown of RASAL2 inhibited LC3-II accumulation or GFP-LC3 puncta formation. In silico analysis of RASAL2 revealed two potential LC3 interacting region motifs (LIR), which could point to an interaction between these two proteins. These data suggest that RASAL2 is involved in autophagy and is regulated by autophagy in a negative feedback manner.

List of scientific papers

I. Pellegrini P, Dyczynski M, Sbrana FV, Karlgren M, Buoncervello M, Hägg- Olofsson M, Ma R, Hartman J, Bajalica-Lagercrantz S, Grandér D, Kharaziha P, De Milito A. Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells. Oncotarget. 2016; 7(24):35703-35723.
https://doi.org/10.18632/oncotarget.9601

II. Dyczynski M, Vesterlund M, Björklund AC, Zachariadis V, Janssen J, Gallart-Ayala H, Daskalakis E, Wheelock CE, Lehtiö J, Grandér D, Pokrovskaja Tamm K, Nilsson R. Metabolic reprogramming of acute lymphoblastic leukemia cells in response to glucocorticoid treatment. Cell Death & Disease. 2018. [Accepted]
https://doi.org/10.1038/s41419-018-0625-7

III. Dyczynski M, Yu Y, Otrocka M, Parpal S, Braga T, Henley AB, Zazzi H, Lerner M, Wennerberg K, Viklund J, Martinsson J, Grandér D, De Milito A, Pokrovskaja Tamm K. Autophagy inhibition by small molecule inhibitors of Vps34 improves sensitivity of breast cancer cells to Sunitinib. Cancer letters. 2018; 435:32–43.
https://doi.org/10.1016/j.canlet.2018.07.028

IV. Dyczynski M, Knopf P, Serviss J, Lerner M, Grandér D, Pokrovskaja Tamm K. Regulation of RASAL2 and RASAL2-AS during autophagy. [Manuscript]

History

Defence date

2018-09-14

Department

  • Department of Oncology-Pathology

Publisher/Institution

Karolinska Institutet

Main supervisor

Pokrovskaja Tamm, Katja

Co-supervisors

De Milito, Angelo

Publication year

2018

Thesis type

  • Doctoral thesis

ISBN

978-91-7831-182-8

Number of supporting papers

4

Language

  • eng

Original publication date

2018-08-24

Author name in thesis

Dyczynski, Matheus

Original department name

Department of Oncology-Pathology

Place of publication

Stockholm

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