R(h)oad to antitumour therapy

Commentary

Autophagy, an evolutionarily intracellular degradation catabolic process maintains cellular homeostasis during stress conditions to recycles nutrients from damaged organelles^{1, 2}. Importantly, although autophagy plays a dual role in tumor promotion and suppression in many cancers, inhibition of autophagy plays a vital role in cancer immunotherapy. Studies have showed that altering the autophagic process by inhibiting or inducing can promote the effectiveness of immunotherapy^{3, 4}. Immunogenic cell death (ICD) is a distinctive immune response induced by anticancer chemotherapeutics resulting in cell death inducted by cellular stress and eventually release of DAMPs (damage-associated molecular patterns)^{5, 6}. Pharmacology inducing ICD activates autophagy in tumor cells, that together with radiotherapy/chemotherapy boosts efficacy by promoting ICD⁷.

In their letter to editor, Yang and collaborators (Yang et al., In press) propose that pharmacological enhancement in the autophagy process can be effective in boosting anticancer immune responses to immunogenic cell death drugs^{8, 9}. They propose that ginsenoside Rh2 (G-Rh2) heightened the MTX (mitoxantrone)-induced ICD including ATP release increase, discharge of HMGB1 (high mobility group box 1) and repositioning of calreticulin (CALR) to the membrane enhancing anti-tumor immune responses (Figure 1). G-Rh2 are ginsenosides, the main components from ginseng and proved to have pharmacological anti-cancer capabilities, inducing autophagy by activating transcriptional factors EB (TFEB) and E3 (TFE3) which adds to the collaborative effect of G-Rh2 that together with the chemotherapy drug MTX activates ATP release^{10, 11}.

Figure 1.

Ginsenoside (G-Rh2) in combination with chemotherapy drug MTX (mitoxantrone) enhance immunogenic cell death (ICD) in cancer cells.

Yang et al. showed that in U2OS cells (human bone osteosarcoma epithelial cells) G-Rh2 is responsible for upregulating LC3-II levels, which is further enhanced by the addition of CQ lysosomal inhibitor, proving that G-Rh2 promotes autophagy in osteosarcoma epithelial cells. They went beyond and using immunofluorescence and western blotting to determine that G-Rh2 increases the expression of TFEB and TFE3. Moreover, once these genes are knockdown Rh2-mediated effect is blocked. The authors also demonstrated that G-Rh2 can induce ICD on different concentrations of MTX, an anti-cancer chemotherapy drug. To better understand how G-Rh2 induces ICD, they showed that low concentrations of MTX together with G-Rh2, decrease intracellular ATP levels (preferentially released); however, once ATG5 gene was knockdown there was a clear inhibition of autophagy. ATG5 is required for autophagy and it is vital for the formation of autophagosomes¹², thereby establishing that autophagy if the fundamental mechanism that needs to be activated for successful cancer therapy using chemotherapy drug.

Interestingly, Yang and collaborators also determined that when inhibiting endoplasmic reticulum (ER) stress with the addition of 4-PBA (4-phenylbutyric acid), lead to the relocation of calreticulin on the cell surface, and the release of HMGB1 (high mobility group box 1) increasing antitumor effects. This is consistent with previous findings where induction of ER stress leads to alarmin release and cell death¹³; however, the reason as why ER stress is induced upon the addition of G-Rh2 needs to be established. The unfolded protein response (UPR) main purpose is to restore the ER’s homeostasis, however a continual UPR activation can trigger cell death pathways^13,14. Additionally, they suggested that the combination of G-Rh2 and MTX also induce apoptosis and it is increased by the lysosomal inhibitor CQ, indicating that the apoptosis pathway is also implicated in antitumor activities. Moreover, Z-VAD-FMK, an apoptosis inhibitor, constrains apoptosis by G-Rh2 together with MTX, but neither influenced intracellular ATP levels, and cell surface calreticulin exposure. This is interesting, and although the authors assume that maybe the anti-tumor effect of G-Rh2 plus MTX is activated by ICD rather than apoptosis, this needs to be further evaluated to differentiate between ICD and apoptosis. One possibility could be that necrosis or other form of immunogenic-induced cell death, as well as activation of specific immune cells could also be initiated in ICD, which needs to be evaluated. Overall, Yang et al. showed that G-Rh2 activates autophagy and induces ER stress by eIF2α and together with MTX enhances ICD, resulting in antitumor effects in mice in vivo. They propose a connection between autophagy induction by G-Rh2-activated by transcriptional factors EB/E3 and ICD promote antitumor effect. Additionally, the authors suggests that G-Rh2 might be a unique drug candidate for improving the antitumor effects of immunogenic chemotherapies.

Although the research findings are interesting and relevant to cancer therapy, additional outstanding questions require further investigations. While G-Rh2 is expected (considering the current knowledge in the field and validate the approach used herein), it is critical to remind that there are many other pathways (such as calcium and cell signaling) that are involved in antitumor activity, which needs to be further studied. Authors only observed ATG5 pathway, although it is a key autophagic factor it belongs to a bigger complex comprised by ATG8 and ATG12¹⁵, it could be of interest to further investigate those markers as well to see if the effects of Rh2 is specific for ATG5 or activate the whole complex. It should be noted that the analysis does support the use of G-Rh2 in conjunction with chemotherapy drugs in anticancer therapy¹⁶, however specific issues can result when making these assumptions on such small group of animals. It would be interesting to see the treatment on a larger group of animals with a control group of autophagic deficient mice to see tumor growth on different treatment concentrations and duration or use PERK and elF2α knockouts mice to further investigate the crosstalk between autophagy and ER stress. Although the use of MTX is a great starting point, a detailed study using additional antitumor agents should be performed to establish if the effect of G-Rh2 is specific or can be used in combination to other chemotherapeutic agents. Finally, identification of immune cells along with H&E staining of infiltrating immune cells on different time points could further expand on this important observation.

Data Availability:

No data is generated in this commentary