Nifuroxazide enhances radiotherapy efficacy against hepatocellular carcinoma by upregulating PD-L1 degradation via the ubiquitin-proteasome pathway

Reviewer #1 (Public Review):

The authors found that nifuroxazide has the potential to augment the efficacy of radiotherapy in HCC by reducing PD-L1 expression. This effect may be attributed to increased degradation of PD-L1 through the ubiquitination-proteasome pathway. The paper provides new ideas and insights to improve treatment effectiveness, however, there are additional points that could be addressed.

-The paper highlights that the combination of nifuroxazide increases tumor cell apoptosis. A discussion regarding the potential crosstalk or regulatory mechanisms between apoptotic pathways and PD-L1 expression would be valuable.

-The benefits and advantages of nifuroxazide combination could be compared to the current clinical treatment options.

Reviewer #2 (Public Review):

Summary:
Zhao et al. aimed to explore an important question - how to overcome the resistance of hepatocellular carcinoma cells to radiotherapy? Given that the immune-suppressive microenvironment is a major mechanism underlying resistance to radiotherapy, they reasoned that a drug that blocks the PD-1/PD-L1 pathway could improve the efficacy of radiation therapy and chose to investigate the effect of Nifuroxazide, an inhibitor of stat3 activation, on radiotherapy efficacy in treating hepatocellular carcinoma cells. From in vitro experiments, they find combination treatment (Nifuroxazide+ radiotherapy) increases apoptosis and reduces proliferation and migration, in comparison to radiotherapy alone. From in vivo experiments, they demonstrate that combined treatment reduces the size and weight of tumors in vivo and enhances mice survival. These data indicate a better efficacy of combination therapy compared to radiotherapy alone. Moreover, they also determined the effect of combination therapy on tumor microenvironment as well as peripheral immune response. They find that combination therapy increases infiltration of CD4+ and CD8+ cells as well as M1 macrophages in the tumor microenvironment. Interestingly, they find that the ratio of Treg cells in spleen is increased by radiotherapy but decreased by Nifuroxazide. Considering the immune-suppressive role of Treg cells, this finding is consistent with reduced tumor growth by combination therapy. However, it is unclear whether the combined therapy affects the ratio of Treg cells in the tumors or not. The most intriguing part of the study is the determination of the effect of Nifuroxazide on PD-L1 expression in the context of radiotherapy. Considering Nifuroxazide is a stat3 activation inhibitor and stat3 inhibition leads to reduced expression of PD-L1, one would expect Nifuroxazide decreases PD-L1 expression through stat3. However, they found that the effect of Nifuroxazide on PD-L1 is dependent on GSK3 mediated Proteasome pathways and independent of stat3, in the given experimental context. To determine the relevance to human hepatocellular carcinoma, they also measured the PD-L1 expression in human tumor tissues of HCC patients pre- and post-radiotherapy. The increased PD-L1 expression level in HCC after radiotherapy is impressive. However, it is unclear whether the patients being selected in the study had resistant disease to radiotherapy or not.

Overall, the data are convincing and supportive to the conclusions.

Strengths:

1. Novel finding: Identified novel mechanism underlying the effect of Nifuroxazide on PD-L1 expression in hepatocellular carcinoma cells in the context of radiotherapy.
2. Comprehensive experimental approaches: using different approaches to prove the same finding. For example, in Fig 4, both IHC and WB were used. In Fig 5, both IF and WB were used.
3. Human disease relevance: Compared observations in mice with human tumor samples.

Weaknesses:
1. It is hard to tell whether the observed phenotype and mechanism are generic or specific to the limited cell lines used in the study. The in vitro experiments were performed in one human cell line and the in vivo experiments were performed in one mouse cell line.
2. The study did not distinguish the effect of increased radiosensitivity by nifuroxazide from combined anti-tumor effects by two different treatments.

Reviewer #3 (Public Review):

Summary:
In this study, the authors embarked on an exploration of how nifuroxazide could enhance the responsiveness to radiotherapy by employing both an in vitro cell culture system and an in vivo mouse tumor model.

Strengths:
The researchers conducted an array of experiments aimed at revealing the function of nifuroxazide in aiding the radiotherapy-induced reduction of proliferation, migration, and invasion of HepG2 cells.

Weaknesses:
The authors did not provide the molecular mechanism through which nifuroxazide collaborates with radiotherapy to effectively curtail the proliferation, migration, and invasion of HCC cells. Moreover, the evidence supporting the assertion that nifuroxazide contributes to the degradation of radiotherapy-induced upregulation of PD-L1 via the ubiquitin-proteasome pathway appears to be insufficient. Importantly, further validation of this discovery should involve the utilization of an additional syngeneic mouse HCC tumor model or an orthotopic HCC tumor model.