RUNX2 Isoform II Protects Cancer Cells from Ferroptosis by Promoting PRDX2 Expression in Oral Squamous Cell Carcinoma

Reviewer #1 (Public Review):

Summary:
In this paper, authors investigated the role of RUNT-related transcription factor 2 (RUNX2) in oral squamous carcinoma (OSCC) growth and resistance to ferroptosis. They found that RUNX2 suppresses ferroptosis through transcriptional regulation of peroxiredoxin-2. They further explored the upstream positive regulator of RUNX2, HOXA10 and found that HOXA10/RUNX2/PRDX2 axis protects OSCC from ferroptosis.

Strengths:
The study is well designed and provides a novel mechanism of HOXA10/RUNX2/PRDX2 control of ferroptosis in OSCC.

Weaknesses:

According to the data presented in (Figure 2F, Figure 3Fand G, Figure 5D and Figure 6E and F), apoptosis seems to be affected in the same amount as ferroptosis by HOXA10/RUNX2/PRDX2 axis, which raises questions on the authors' specific focus on ferroptosis in this study. Reasonably, authors should adapt the title and the abstract in a way that recapitulates the whole data, which is HOXA10/RUNX2/PRDX2 axis control of cell death, including ferroptosis and apoptosis in OSCC.

Comments:

- In the description of the result section related to Figure 3E, the author wrote "In addition, we found that isoform II-knockdown induced shrunken mitochondria with vanished cristae with transmission electron microscopy (Figure 3E). These results suggest that RUNX2 isoform II may suppress ferroptosis." The interpretation provided here is not clear to the reviewer. How shrunken mitochondria and vanished cristae can be linked to ferroptosis?
- The electron microscopy images show more elongated mitochondria in the RUNX2 isoform II-KO cells than in RUNX2 isoform II positive cells, which might result from the fusion of mitochondria. These images should completed with a fluorescent mitochondria staining of these cells.
- What is the oxygen consumption rate in RUNX2 KO cells?
- The increase in cell proliferation after RUNX2 overexpression in Figure 2A is not convincing, is there any differences in their migration or invasion capacity?
- The in vivo study shows 50% reduction in primary tumor growth after RUNX2 inhibition by shRNA in CAL 27 xenografts, but only one shRNA is shown. Is this one shRNA clone? At least 2 shRNA clones should be used.
- Apoptosis and necroptosis seem to be affected in the same amount as ferroptosis by HOXA10/RUNX2/PRDX2 axis. This is evident from experiments in Figure 3E, F and from Figure 6E, F and Figure 3G. Either Fer-1, Z-VAD,or Nec-1 used alone, were not able to fully restore cell proliferation to control cell level, which implies an additive effect of ferroptosis, apoptosis and necrosis. The author should verify potential additive or synergistic effect of the combination of Fer-1 and Z-VAD in these assays after si-RUNX2 in Figure 3 F and G and after si-HOX assays.
- What is the effect of PRDX2 or HOXA10 depletion on tumor growth?
- What is the clinical relevance of HOXA10 in OSCC patients?

Reviewing #2 (Public Review):

This paper reports the role of the Isoform II of RUNX2 in activating PRDX2 expression to suppress ferroptosis in oral squamous cell carcinoma (OSCC).
The following major issues should be addressed.

A major postulate of this study is the specific role of RUNX2 isoform II compared to isoform I.

Figure 1F shows association between patient survival and Iso II expression, but nothing is shown for Iso I, this should be added, in addition the number of patients at risk in each category should be shown.
The authors test Iso I and Iso II overexpression in CAL27 or SCC-9 model cell lines. In Fig. 2A in CAL27, the overexpression of Iso II is much stronger than Iso I so it seems premature to draw any conclusions. More importantly, however, no Iso I silencing is shown in either of the cell lines nor the xenografted tumours. This is absolutely essential for the authors hypothesis and should be tested using shRNA in cells and xenografted tumours.

A major conclusion of this study is that Iso II expression suppresses ferroptosis. To support this idea, the authors use the inhibitor Ferrostatin-1 (Fer-1). While Fer-1 typically does not lead to a 100% rescue, here the effect is only marginal and as shown in Figures 3F and G only marginally better than Z-VAD or Necrostatin 1. These data do not support the idea that the major cause of cell death is ferroptosis. Instead, Iso II silencing leads to cell death through different pathways. The authors should acknowledge this and rephrase the conclusion of the paper accordingly.
Moreover, the authors consistently confound cell proliferation with cell death.

In Fig. 4A the authors investigate GPX1 expression, whereas GPX4 is often the key ferroprosis regulator, this has to be tested. This is important as the authors also test the effect of the GPX4 inhibitor RSL3, however, the authors do not determine IC50 values of the different cell lines with or without Iso II overexpression or silencing or compared to other RSL3 sensitive or resistant cells. Without this information, no conclusions can be drawn.

In summary, while the authors show that RUNX2 Iso II expression enhances cell survival, the idea that cell death is principally via ferroptosis is not fully established by the data. The authors should modify their conclusions accordingly.