Schweinfurthins And Immunogenic Cell Death
Open Access
- Author:
- Zhang, Ruoheng
- Graduate Program:
- Biomedical Sciences (PHD)
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- October 20, 2023
- Committee Members:
- Jong Yun, Major Field Member
Hong Zheng, Major Field Member
Todd Schell, Outside Unit & Field Member
Raymond Hohl, Chair & Dissertation Advisor
Jeffrey Neighbors, Major Field Member
Lisa Shantz, Program Head/Chair - Keywords:
- Schweinfurthins
Immunogenic Cell Death (ICD)
calreticulin (CRT)
ER stress
apoptosis - Abstract:
- Over the past few decades, the emergence of immunotherapy strategies has revolutionized cancer treatment. Immune checkpoint inhibition is one strategy in which pharmacologic administration of immune checkpoint inhibitors (ICIs) breaks the inhibitory interactions between tumors and immune cells. This results reactivation of the immune system to recognize and attack tumor cells. The combination of chemotherapeutics with immunotherapy, especially the ICIs, has been shown to increase the anti-cancer activities of ICIs. The induction of immunogenic cell death (ICD) may contribute to this effect. ICD is a form of regulated cell death wherein the dying cancer cells activate the immune system and elicit anti-cancer immune responses. Cells undergoing ICD release danger-associated molecular patterns (DAMPs) which allow the tumor cells to be recognized and taken up by professional antigen-presenting cells known as dendritic cells (DCs). Activated DCs present tumor-antigen to naïve T cells and activate them to become cytotoxic CD8+ T cells. Finally, CD8+T cells specifically target and eliminate the tumor cells. Therefore, the release of DAMPs is crucial for ICD and is considered to be a detectable marker of ICD. The translocation of chaperone protein calreticulin (CRT) from the endoplasmic reticulum (ER) to the plasma membrane is a key DAMP that serves as an “eat me” signal to attract DCs to uptake tumor cells. The mechanism of CRT exposure induced by ICD inducers such as anthracyclines has been summarized in a “canonical pathway”. There are three hierarchical modules in this pathway, including: (i) “ER stress module,” which involves PERK activation and its-induced eIF2a phosphorylation, ROS and/or NO production; (ii) “apoptotic module,” which involves caspase-8, cleavage of Bap31 and activation of the Bcl-2 family proteins BAX/BAK;(iii) The “translocation module,” which involves transport of CRT from ER to plasma membrane. CRT first anterogradely transport from ER lumen to Golgi via actin cytoskeleton rearrangement, and then exocytotic vesicles move up and fuse with the plasma membranes via the interaction of the vesicle-associated SNARE (VAMP1) and the plasma membrane–associated SNARE (SNAP23). Schweinfurthins are a family of compounds that display differential cytotoxicity against human cancer cell lines. The first schweinfurthin was identified in 1992 from the leaves of the Cameroonian flowering tree Macaranga schweinfurthii. The first tetracyclic stilbene analog of the natural schweinfurthins 3-deoxyschweinfurthin-B (3dSB) was synthesized in 2005. A previous animal study revealed that schweinfurthin analogs TTI-3114 (MeSG) and TTI-4242 induce transient B16F10 tumor regression in C57BL/6 mice. Its combination with anti-PD-1 ICI not only enhanced the anti-tumor effect of anti-PD-1 antibody in the B16F10 murine melanoma model but also provoked durable, protective anti-tumor immunity. These effects require a fully competent immune system. However, we do not know the mechanisms behind these findings. We hypothesized that schweinfurthins may induce ICD to enhance the anti-tumor effect of anti-PD-1. Here we show that schweinfurthin analog MeSG induces significant cell surface calreticulin (Ecto-CRT) exposure in a time- and concentration-dependent manner. Analog TTI-4242-treated B16F10 cells increased tumor cell phagocytosis by immature dendritic cells (iDCs) and activated these iDCs to mature. Interestingly, this CRT exposure differs from the canonical pathway in several aspects. MeSG does not cause ER stress and does not require PERK to induce CRT exposure. Caspase 8 inhibitors partially rescued cells from MeSG-induced apoptosis, but failed to reduce Ecto-CRT level. MeSG did not cause ERp57 exposure and the absence of ERp57 expression did not reduce Ecto-CRT. Finally, an intact ER to Golgi transport system is required for this phenomenon. Our study reveals that schweinfurthins cause CRT exposure on tumor cells and schweinfurthin-treated B16F10 cells can activate DCs in vitro. This is the first time that the effect of schweinfurthin on immune cells has been assessed. These findings provide insight into what was observed in the animal study and provide the rationale for future in vivo studies. The mechanism of CRT exposure is as yet not fully explored and the involvement of a non-canonical pathway needs to be further elucidated. These results lend support the development of the schweinfurthin compounds as drugs to enhance clinical response to immunotherapy and highlight the need for additional research on the mechanisms of ICD induction by MeSG