Dysregulation of Type I Interferon (IFN-I) Signaling: A Potential Contributor to Racial Disparity in Hepatocellular Carcinoma (HCC)
, Milton Omar Faison 7, Rafat Ali Siddiqui 2,* and Devanand Sarkar 8,*Round 1
Reviewer 1 Report
In this study the authors showed that IFN type 1 signaling is involved in hepatocellular carcinoma, especially in African American. The authors showed that IFN signaling is more in AA than white American , and more signaling in HCV infected ones. Using Ginger, they showed that it could downregulate the IFN signaling , especially in cell lines derived from AA.
Major Points
1) HCV diagnosis should be described in details to know the status of HCV positivity: active, past, or occult HCV infection.
2)ALl WB original uncropped blots should be uploaded as supple.
3) FOR IHC, WB, quantification of band intensity and/or staining signaling should be performed.
4) Since the authors collect blood and tumor tissues. Did the authors see the same trend of DEGs among PBMCs and hepatocytes?
5) Figure 5A: add scale bar. And I do not see the significance of this part.
Moderate language editing
Author Response
We thank the reviewers for their constructive criticisms and finding our manuscript important and relevant. A point-by-point response is provided below:
Reviewer#1
1) HCV diagnosis should be described in details to know the status of HCV positivity: active, past, or occult HCV infection.
Answer: We now provide the information in Table 2 using an asterisk which indicates presence of HCV at the time of HCC diagnosis. All other patients had history of HCV infection but HCV was not detected at the time of HCC diagnosis.
2) All WB original uncropped blots should be uploaded as supple.
Answer: We thank the reviewer for the suggestion. We have now added the original blots in the supplement.
3) FOR IHC, WB, quantification of band intensity and/or staining signaling should be performed.
Answer: We thank the reviewer for the suggestion. Quantification of Western blot was presented in original Figure 6B. We now include quantification of RNA in-situ hybridization for IFI6 and IHC for OAS1 and MX1 in Figures 3C and 4C in the revised manuscript.
4) Since the authors collect blood and tumor tissues. Did the authors see the same trend of DEGs among PBMCs and hepatocytes?
Answer: This is an excellent suggestion. Tissue and Data Acquisition and Analysis Core (TDAAC) facility of Virginia Commonwealth University has recently started collecting both tumor tissues and blood. Many of the samples used in this study are archived tumor tissues, especially from AA patients, which were collected at a time point when blood collection was not routine. As such it was not possible to perform correlation analysis of DEGs in PBMCs. We will follow up with the reviewer’s suggestion and check whether ISG signature can be used as a blood biomarker in PBMC in future studies.
5) Figure 5A: add scale bar. And I do not see the significance of this part.
Answer: We apologize for the omission and now have added the scale bar in Fig. 5E.
Reviewer 2 Report
In this submission, the authors demonstrate that IFN induced genes are more highly expressed in HCC tumors from AA/black patients compared to white patients. Furthermore similar differences were observed in cell lines derived from patients. Racial disparity in cancer is a very important topic and this paper is most relevant to that important issue. I do have a few comments that could be considered.
1. Evidence is growing that an IFN signature correlates with endogenous retrovirus gene expression. Has this been evaluated in the different samples?
2. Are there any differences in IFN receptor gene expression?
3. Although the authors report no difference in IFN gene expression did they look for Type III IFN? Probably unlikely especially due to the lack of increased pSTAT1.
4. If the white cell lines are treated with IFN, do the genes studied increase in expression? Do the AA/blck lines further increase expression?
5. Have different batches of ginger extract been compared? If so, are consistent dose response curves observed?
6. The concentrations of ginger extracts tested seem pretty high. Could such concentrations be obtained in vivo or would they be toxic to a host (I appreciate this may not have ever been tested).
Author Response
We thank the reviewers for their constructive criticisms and finding our manuscript important and relevant. A point-by-point response is provided below:
Reviewer#2
- Evidence is growing that an IFN signature correlates with endogenous retrovirus gene expression. Has this been evaluated in the different samples?
Answer: Endogenous retroviral genes have been shown to be overexpressed in HCC and promote the disease. The reviewer’s suggestion to check correlation between ISGs and ERV is highly relevant. There are numerous endogenous ERV genes. We randomly selected a few genes from different ERV groups, namely, ERV3-1, ERVH48-1, ERVK3-1, ERVK13-1, ERVK9-11, ERVW-1 and ERVFRDE1. We checked correlation between the ISGs we identified, OAS1, ISG15, MX1 and IFI6, and the aforementioned ERV genes in TCGA and in our in-house RNA-seq data. We observed no correlation among any of these genes suggesting that ERVs may not dictate overexpression of ISGs in AA HCC patients.
- Are there any differences in IFN receptor gene expression?
Answer: Both white and AA HCC patients express receptors for interferon alpha/beta (IFNAR1 and IFNAR2), interferon gamma (IFNGR1 and IFNGR2) and IL-10 (IL10RA and IL10RB). However, there is no difference in their expression levels between white and AA HCC patients.
- Although the authors report no difference in IFN gene expression did they look for Type III IFN? Probably unlikely especially due to the lack of increased pSTAT1.
Answer: As acknowledged by the reviewer we did not find any difference in Type III IFN levels between white and AA HCC patients.
- If the white cell lines are treated with IFN, do the genes studied increase in expression? Do the AA/black lines further increase expression?
Answer: For our studies we used HepG2 cell from white HCC patient and Hep3B cell from AA HCC patient. Both these cells are responsive to type I IFN resulting in induction of ISGs. This is observed not only in our hands but existing literature also shows type I IFN responsiveness of these cells.
- Have different batches of ginger extract been compared? If so, are consistent dose response curves observed?
Answer: Yes, the horticulture scientists at VSU are cultivating ginger in high tunnels with controlled environmental conditions. In addition, we used baby ginger in these experiments which are harvested at a fixed time after their sprouting, and we do not find variations in the polyphenolic contents in different batches.
- The concentrations of ginger extracts tested seem pretty high. Could such concentrations be obtained in vivo or would they be toxic to a host (I appreciate this may not have ever been tested).
Answer: Yes, we agree with the reviewer. In the present study we have used crude extract. Ginger extract concentration in similar range has previously been used by other investigators (for example: Akimoto M, Iizuka M, Kanematsu R, Yoshida M, Takenaga K (2015) Anticancer Effect of Ginger Extract against Pancreatic Cancer Cells Mainly through Reactive Oxygen Species-Mediated Autotic Cell Death. PLoS ONE 10(5): e0126605. https://doi.org/10.1371/journal.pone.0126605). We anticipate that the active principal component responsible for anti-cancer effect may be present in ng concentration.
Round 2
Reviewer 1 Report
No further comments
Moderate language editing
