MicroRNA-203 represses selection and expansion of oncogenic Hras transformed tumor initiating cells

1) The authors should simplify their presentation of the genomic data and be more strategic about which datasets they include in a revised manuscript, including only those datasets and bioinformatic analyses that offer clear mechanistic insight.

We thank you for the suggestion. We have now revised Figure 6, in which we used an extensive set of genomic analyses to interrogate the impact of miR-203 on the transcriptome and identify high-confidence miR-203 targets. Specifically, we moved Figure 6D, E, F, I to Figure 6–figure supplements 1 and 4. These data were utilized to synthesize our conclusion. By relocating them to figure supplements, we simplified our main Figure 6 to present the results of our transcriptome analysis and HITS-CLIP analysis for identifying miR-203 targets. In addition, we added another Figure 6–figure supplement 3 to document the strong overlap between Ago2 footprint and miRNA seed matches and list all detected miRNA seed matches detected in our experiments. These data further validated our HITS-CLIP study and provided additional data for readers, who may be interested in miRNA targets in keratinocytes.

2) More mice should be analyzed in Figure 3E-G, especially at the E16 time-point when only 2 embryos were examined. The trend towards increased BrdU/EdU incorporation may then become significant, which would be an important finding since this is the only potential phenotype in the intact knockout animals. With the currently presented data, it is an overstatement to claim that “miR-203 functions as a gatekeeper to limit cell division when the proliferation rate is high during early embryonic skin development…” (the subsection “Genetic deletion of miR-203 impacts early epidermal development in mouse”, second paragraph).

During our revision, we have set up more than 6 breeding pairs in an effort to produce miR-203 KO embryos. In total, we have generated one pair of E16 WT and KO embryos, and one pair of E17 WT and KO embryos because of the small litter size and our heterozygous to heterozygous breeding strategy, which has 25% chance to produce KO. Nevertheless, we incorporated these new embryonic data to our existing data in Figure 3F. We now have three pairs of E16 embryos and four pairs of E17 pairs. The p-value for the E16, BrdU/EdU incorporation study is 0.07 and the p-value for the E17, BrdU/EdU incorporation study is 0.13. In addition, the p-value for the E16 epidermal thickness study is 0.03 and the p-value for the E17, epidermal thickness study is 0.01. The new data are consistent with our original observations, that is miR-203 KO epidermis are more proliferative than the WT counterparts. Together, these data further strengthened our original observations.

We also revised our statement to “miR-203 limits cell division when the proliferation rate is high during early embryonic skin development”, to more accurately reflect the observed defects.

3) Where are the data that show that “genes involved in regulation of mitotic cell cycle, DNA synthesis, and metabolism were prominently downregulated upon miR-203 induction” (the subsection “Comprehensive identification of miR-203 targets reveals regulation of the Ras signaling pathway”, second paragraph)? Was an unbiased method such as GSEA or gene ontology employed?

These statements were based on unbiased gene ontology analyses, which were presented in the panel of “miR-203 inducible analysis” in “Figure 6–source data 1_GO analysis.” However, we didn’t link this source in our original manuscript and we apologize for the negligence. In the revised manuscript (the subsection “Comprehensive identification of miR-203 targets reveals regulation of the Ras signaling pathway”, third paragraph), we stated “GO-analysis demonstrated that genes involved in regulation of mitotic cell cycle, DNA synthesis and metabolism were prominently downregulated upon miR-203 induction (Figure 6–source data 1).”

4) Based on the data presented in this manuscript, it is an overstatement to claim that “miR-203 directly targets the Ras-mediated signaling pathway” (end of the subsection “Comprehensive identification of miR-203 targets reveals regulation of the Ras signaling pathway”). At best, a few targets that contribute to multiple pro-proliferative pathways were identified. It is plausible that miR-203 exerts its effects via the coordinated, although minor, downregulation of a broad set of targets, some of which include members of the Ras pathway.

We agree with the reviewers’ assessment. Based on our data and the reviewers’ suggestion, we revised our conclusion (in the subsection “Comprehensive identification of miR-203 targets reveals regulation of the Ras signaling pathway”) to “This collection of miR-203 targets suggests that miR-203 targets multiple pathways, including several components of the Ras signaling pathway, to suppress cell proliferation.”

5) shRNA knockdown experiments with putative targets (Figure 7F-G) should be performed in Hras-transformed miR-203 +/+ and -/- cells to see if these targets are relevant to transformation in these settings.

We carried out the shRNA knockdown experiments for both Pola1 and Hbegf in HRas-transformed miR-203 WT and KO keratinocytes as suggested by the reviewers. In both cases, we observed strong reduction in colony forming capacity of these transformed cells, similar to our original observations with non-transformed keratinocytes. Collectively, these data further support the requirement of Pola1 and Hbegf in cell proliferation/survival of both HRas transformed and non-transformed cells. We present these data as Figure 7–figure supplement 2 to in the revised manuscript.