Interrelationship between autophagy and ER stress, and their impact on the fate of prostate cancer cells treated with therapeutic drugs

A problem in cancer therapy is resistance to treatment that is intended to kill the cancer cells or inhibit their growth. It is thus important to understand how therapeutic drugs affect cellular stress responses that influence the cancer cell survival and growth, and the relationship between them. Endoplasmic reticulum stress (ER stress) and autophagy (lysosomal degradation of intracellular material) are two cellular stress responses which often appear to be affected by cancer therapy, and which likely play an important role in determining the fate of chemotherapy-treated cancer cells. In this project we investigated how different therapeutically relevant compounds affect ER stress and autophagic activity in prostate cancer cells and other cancer cell types, how ER stress responses affect autophagic activity, and how ER stress and autophagy affect cancer cell survival and growth. Results summary: 1) Important foundational and methodological work: To our surprise, and contrary to what has been believed for the last 10-15 years, we found that the autophagy marker LC3 is not required for general autophagy in mammalian cells, and LC3 transport to the cellular degradation machinery (lysosomes) failed to reflect autophagic activity (Exp Cell Res 333, 21-38, 2015; FEBS J 282, 2202-14, 2015; Autophagy 12, 439-41, 2016). This is a very important finding because the knowledge about the biological and physiological roles of autophagy in general, and about the relationship between ER stress and autophagy in cancer cells, is largely based on the use of LC3 as a marker of autophagy, rather than on direct measurements of autophagic activity. Consequently, it was essential for us to use LC3-independent methods to measure autophagy. We succeeded in transforming and validating two originally laborious biochemical methods into time- and cost-effective assays to measure autophagic activity (Methods 75, 25-36, 2015; Autophagy 12, 1-222, 2016; Methods Enzymol 587, 351- 364, 2017; JoVE, 127, 2018; Bio-Protoc, 8, 2018). 2) With these improved methods, we conducted a detailed study on how autophagy and cell death in cancer cells are affected by the ER stress-inducing drugs tunicamycin (glycosylation inhibitor), thapsigargin (ER calcium pump inhibitor), and therapeutically relevant analogs of the latter, two of which are under clinical trials. - Thapsigargin and clinically relevant thapsigargin analogs blocked autophagy in prostate- and other cancer cells. Moreover, they induced a prolonged ER stress response that led to cell death. These effects were due to a severe reduction in ER calcium, but not to changes in cytosolic calcium levels. At lower concentrations, the drugs induced a partial depletion of ER calcium. This did not result in ER stress or cell death, and did not affect autophagy, but had a clear growth inhibitory effect on cancer cells. This is of general interest since ER stress and changes in ER calcium levels often are linked. Moreover, it suggests that different doses of the clinically relevant thapsigargin analogs may give different cellular effects, all of which will help to stop cancer cell proliferation. The results are published in two papers (J Biol Chem 292, 19656-73, 2017; Cell Calcium 76, 48-61, 2018). - We have investigated in more detail how thapsigargin and the analogs induce cell death. We found that the drugs induce a programmed cell death via transcriptional upregulation of pro-apoptotic gene products. Furthermore, we have uncovered which parts of ER stress signaling convey the death signals, as well as the role of autophagy and autophagy-related gene products. The findings are under preparation for publication. - Unlike thapsigargin and the analogs, we found that tunicamycin activated autophagy in prostate cancer cells, and in a manner that was totally dependent on ER stress signaling. Tunicamycin did not reduce ER calcium levels, indicating that ER stress is not always associated with changes in calcium homeostasis. Two components of ER stress signaling were essential for ER stress-induced autophagy. These components influenced two different steps in the autophagic process via transcriptional effects (changes in gene expression) and signaling, respectively. The findings are under preparation for publication. Tunicamycin could induce autophagy equally well in non-tumorigenic cells as in cancer cells. However, tunicamycin did not activate autophagy in all cell types, despite universal induction of ER stress signaling. Taken together, the project has given us a new understanding of how the different parts of ER stress signaling regulate autophagy and cell death in cancer cells exposed to therapy-related stress. Moreover, the results indicate that the effect of ER stress on autophagy, cell death and growth is highly context-dependent. Understanding why and which factors determine the outcome under different conditions will be important in defining new effective combinations of chemotherapy.

The ability of cancers to resist various types of therapeutic drugs is a major obstacle to effective treatment. Recent studies have implicated endoplasmic reticulum (ER) stress pathways and the lysosomal-degradative pathway of autophagy in mediating respo nses to therapeutic stress. However, this is based on circumstantial evidence since assays to measure autophagic activity have not been used. In the current proposal we will for the first time introduce assays of autophagic sequestration and degradation, which we have recently established in prostate cancer cell lines, in a focused screen of therapeutically relevant drugs for prostate cancer. We will compare prostate cancer cells versus normal prostatic epithelial cells for their ability to activate autop hagy and ER stress pathways in order to assess whether cancer cells may be primed to respond with these pathways upon therapeutic insults. Next, we will use siRNA silencing as well as novel and potent inhibitors of the ER stress pathway to determine the r elationship between ER stress and autophagy in the context of drug-induced stress, and conversely we will measure the effect on ER stress pathways upon selective inhibition of autophagy. Finally, we will determine the effect of alterations in ER stress an d autophagy pathways on cell proliferation and cell death, with the goal of deciphering their impact on cell fate, as well as to define novel drug combinations that can inhibit growth and induce cell death in prostate cancer cells.