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Strand-specific in vivo screen of cancer-associated miRNAs unveils a role for miR-21∗ in SCC progression

Nature Cell Biology volume 18, pages 111–121 (2016)Cite this article

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Abstract

MicroRNAs play diverse roles in both normal and malignant stem cells. Focusing on miRs and/or miR∗s abundant in squamous cell carcinoma (SCC) stem cells, we engineer an efficient, strand-specific expression library, and apply functional genomics screening in mice to identify which of 169 cancer-associated miRs are key drivers in malignant progression. Not previously linked functionally to cancer, miR-21∗ was the second top hit, surfacing in >12% of tumours. miR-21∗ also correlates with poor prognosis in human SCCs and enhances tumour progression in xenografts. On deleting the miR-21 gene and rescuing each strand separately, we document the dual, but independent, oncogenicity of miR-21 and miR-21∗. A cohort of predicted miR-21∗ targets inversely correlate with miR-21∗ in SCCs. Of particular interest is Phactr4, which we show is a miR-21∗ target in SCCs, acting through the Rb/E2F cell cycle axis. Through in vivo physiological miR screens, our findings add an interesting twist to an increasingly important oncomiR locus.

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Figure 1: miRNA profiling reveals complex in vivo miRNA landscapes in stem cells under homeostasis and tumorigenesis.
Figure 2: In vivo tool small accurate (SA)-miR for efficient and faithful miRNA expression.
Figure 3: In vivo pooled screen with the SA-miRNA library identifies candidate oncomiRs in skin SCCs.
Figure 4: In vivo mouse tumorigenesis validates candidate oncomiRs including miR-21∗ in driving SCC progression.
Figure 5: miR-21∗ is required to sustain tumour growth independent of miR-21.
Figure 6: miR-21∗ is physiologically relevant in human SCCs.
Figure 7: miR-21∗ directly targets Phactr4 and regulates tumorigenesis through the Rb/E2F axis.
Figure 8: Phactr4 is a main mediator of oncomiR-21∗ activity in vivo.

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  • 10 December 2015

    In the version of this Article originally published online, the third sentence of the abstract should have read “Not previously linked functionally to cancer, miR-21* was the second top hit, surfacing in >12% of tumours”. This is now correct in all versions of the Article.

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Acknowledgements

We especially thank D. Schramek for intellectual input into the design of the allograft assay. We also thank D. Schramek and A. Sendoel for advice regarding data presentation; J. Racelis for technical assistance; A. Aldeguer, L. Polak, J. Levorse, S. Hacker, M. Sribour, D. Oristian and N. Stokes for assistance with mouse handling and experiments; and Z. Shen, N. Oshimori, S. Luo, K. Lay, M. Kadaja, B. Keyes, A. Asare, M. Laurin, R. Adam and A. Kulukian for helpful discussions. We thank the Comparative Bioscience Center (AAALAC accredited) for care of mice in accordance with National Institutes of Health (NIH) guidelines; Rockefeller Genomics Resource Center (C. Zhao, Director) and Weill Cornell Genomics Resource Center (J. Xiang, Director) for sequencing; Flow Cytometry facility (S. Mazel, Director) for FACS sorting. E.F. is an Investigator of the Howard Hughes Medical Institute. Y.G. is the recipient of a Women & Science Postdoctoral Fellowship, and a Department of Defense Breast Cancer Postdoctoral Fellowship.

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Author notes

  1. Liang Zhang

    Present address: Present addresses: SIBS (Institute of Health Sciences)—Changzheng Hospital Joint Center for Translational Research, Institutes for Translational Research (CAS-SMMU), Shanghai 200025, China; Key Laboratory of Stem Cell Biology, Institute of Health Sciences, SIBS, Chinese Academy of Sciences, Shanghai JiaoTong University School of Medicine, Shanghai 200025, China; Collaborative Innovation Center of Systems Biomedicine, Shanghai 200025, China (L.Z.).,

  2. Yejing Ge and Liang Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Howard Hughes Medical Institute and Laboratory of Mammalian Cell Biology and Development, Rockefeller University, New York, New York 10065, USA

    Yejing Ge, Liang Zhang, Maria Nikolova & Elaine Fuchs

  2. Department of Genetics and Genomic Sciences, Mt. Sinai School of Medicine, New York, New York 10029, USA

    Boris Reva

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Contributions

Y.G. and E.F. designed the experiments and wrote the manuscript. L.Z. designed and characterized the SA-miR vector. Y.G. and L.Z. performed the miR sequencing on FACS-sorted cells and made the lentiviral SA-miR constructs (Figs 1a and 2). Y.G. performed all other experiments and analyses (Figs 1b, c and 3–8). B.R. carried out the TCGA data analyses on miR-21 and miR-21∗ targets. M.N. assisted in CRISPR/CAS of Phactr4. All authors provided intellectual input, and vetted and approved the final manuscript.

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Correspondence to Elaine Fuchs.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 miRNA profiling of skin progenitors from different stages of development, homeostasis and tumourigenesis.

(a) FACS scheme for normal stem cells and progenitors at different stages of skin development, and for papilloma and SCC progenitors and their progenies at different stages of malignant progression. Please refer to Methods for detailed gates. (b,c) Quantitative PCR to quantify the relative mRNA levels for a group of cell-type specific markers to validate the purity of FACS isolated populations: Lhx2 and Pdzrn3 for HF progenitors (E17, P0), Cd34 and Lhx2 for adult HF stem cells, Trp63 for E17 Epidermal progenitors, Sca1 for P0 and adult epidermal progenitors; Ccnd1, Cd44, Krt6 and Vim for SCC-SC upregulated markers and Krt10, Lhx2 and Nfatc1 for SCC-SC downregulated markers compared to HF-SCs. Paired t-tests were used. At least three biological replicates were performed; shown are mean ± s.d. n = 3 independent experiments. ∗P < 0.05.∗∗P < 0.01. (d) SCC signature miRNAs (cut off 100 RRMM) that are conserved between mouse and human and exhibit ≥2X change in basal cells (BCs) of SCCs relative to normal SCs (DEseq P value < 0.05). (e) qPCRs confirmed a subset of miRNA signatures discovered in miRNA profiling. Paired t-tests were used. At least three biological replicates were performed; shown are mean ± s.d. n = 3 independent experiments. ∗P < 0.05.∗∗P < 0.01. (f) miRNA in situ hybridizations of known and SCC-related miRNAs in development and malignant transformation. These results showed that miR-205, an essential miRNA known to govern skin epithelium homeostasis, is broadly and abundantly expressed throughout skin development and transformation; miR-203, known to promote skin stratification and suppress metastasis, is specifically enriched in the suprabasal layers at all stages; miR-125b known to maintain normal SC self-renewal and drive skin tumourigenesis is transiently upregulated during early stage of benign tumourigenesis; miR-744 is broadly expressed during embryogenesis and later becomes more restricted to suprabasal layers during adult and tumour development. At least three biological replicates were performed; shown are representative images. Scale bar = 50 μm.

Supplementary Figure 2 Design and characterization of lentiviral SA-miRNA expression vectors.

(a) Schematic to show differences in miR-21 and miR-21∗ sequences and the corresponding strand-specific SA-miRs.(b) SA-miRNAs are pooled and packaged into lentivirus to transduce cultured mouse SCC cells. Cells were isolated 2 days post infection, sorted with endogenous GFP, and subjected to deep sequencing. The normalized reads for position 1 and 2 isomiRs were plotted to demonstrate faithful 5’ end processing using SA-miRNA system. Grey bar represents control cells transduced with SA vector driving the expression of a scrambled sequence alone, reflecting endogenous isomiR levels. A different Scrambled sequence was used from the SA-Scr shown in the graph. (c) To compare the efficacy of our SA-miRs with oft-used SH-miRs (pENTR/pSM2 (U6), Addgene 17387)40, we performed luciferase reporter assays to examine the effects of miR-125b and miR-203 on their established target genes Scarb1 and p6310,41, respectively, expressed in either the SH-miR or the SA-miR backbone. All vectors were co-transfected with miR mimics at equal concentration in controls and experiments. Similar sensor-mediated inhibition (achieved by anti-sense sequences of miR-125b or miR-203) was observed from both, but stronger target inhibition was observed only from SA-miRNA mediated miR-125b and miR-203 expression when using 3’UTR reporters. Paired t-tests were used to compare SH-miR to SH-control, and SA-miR to SA-control, respectively. At least three biological replicates were performed; shown are mean ± s.d. n = 3 independent experiments. ∗∗P < 0.01. N.S. Not Significant.

Supplementary Figure 3 More candidate oncomiRs in skin SCCs identified from in vivo pooled screen with SA-miRNA library.

(a) Caspase 3 and Ki67 staining of tumours show unchanged apoptosis and heightened mitosis, respectively, in oncogenic HRas tumours derived from the SA-miRNA library transduced cohort compared to that of SA-Scr library. Scale bar = 50 μm. At least five biological replicates were performed; shown are representative images. Ki67 positive nuclei as a percentage of total nuclei was quantified and shown. Paired t-tests were used to compare SA-miR to SA-Scr. Shown are mean ± s.d. n = 5 independent experiments. ∗∗P < 0.01). (b) Targeted deep sequencing of SA-miRNA inserts in the tumours emerging from animals transduced with the SA-miR library showed that three different miRNA families had miRs that were overrepresented in tumours. OncomiRs miR-125b and miR-205 were also captured. Clonal dominance was not observed in regions of the backskin adjacent to the tumour (shown) or in E12.5 or P50 skin (see Fig. 3). Numbers of tumours are shown in parentheses. The frequency of clonal dominance in these tumours was significantly less than that seen for miR-21 and miR-21∗ (see Fig. 3).

Supplementary Figure 4 Validation of miR-21∗ in driving SCC progression in vivo.

(a) Strand-specific expression of SA-miR-21 and SA-miR-21∗ vectors in vivo. Note that SA-miR-21 elevated miR-21 but did not affect miR-21∗ levels; SA-miR-21∗ elevated miR-21∗, but did not affect miR-21 levels in the skin epidermis 3 days after infection. Paired t-tests were used to compare SA-miR to SA-Scr. At least three biological replicates were performed; shown are mean ± s.d. n = 3 independent experiments. ∗P < 0.05,∗∗P < 0.01. (b) In situ hybridizations on day 7 post-wound mouse backskin, showing specific miR-21 and miR-21∗ probe signals in heterozygous but not miR-21-/- skin. MiR-21 locus was transcriptionally activated during wounding15 and has been proposed to be regulated by a number of transcription factors in the context of normal skin and cancer42. Scale bar = 50 μm. (c) SCs purified from the bulge of HFs from WT skin and then transduced with either SA-Scr, SA-miR-21 or SA-miR-21∗ were subjected to colony formation assays in culture. Colonies were stained with Rhodamine B, 2 weeks after seeding at the concentrations indicated at left. Numbers of holoclones (defined by clones >20 μm in diameter and typical of stem cells) are shown in the graph below. Note elevation in the number of stem cells, particularly when miR-21∗ is elevated. (d) SA-miR overexpression of miR-21 or miR-21∗ in keratinocytes resulted in their increased proliferation (top graph) and decreased apoptosis in suspension cultures (bottom graph). Paired t-tests were used to compare SA-miRs to SA-Scr. At least three biological replicates were performed; shown are mean ± s.d. n = 3 independent experiments. If not associated with passenger strand miR, ∗ denotes statistical significance. ∗P < 0.05,∗∗P < 0.01.

Supplementary Figure 5 CRISPR/CAS strategy to knockout miR-21 locus.

Surveyor assay: the CRISPR/CAS-edited mir-21 locus is amplified by PCR from ΔSCC genomic DNA and then digested by an endonuclease enzyme that only cuts the non-matched DNA position (introduced by CRISPR editing). If editing is successful, the 718 bp PCR product is cleaved into 462 and 256 bp smaller bands.

Supplementary Figure 6 miR-21∗ expression and CRIPSR/CAS in human SCCs.

(a) miRNA sequencing data of SCCs show that miR-21∗ but not miR-31∗ or miR-205∗ is stabilized in mouse skin SCCs and human HNSCCs, even though all of these miR guide counterparts are abundant in these tumours. Shown are mean ± s.d. n = 3 independent experiments. (b) In situ hybridization of miR-21 and miR-21∗ in paraffin-fixed human tissue arrays with HNSCCs and normal tissues. Note that hybridizations are not as elegant as with frozen sections (see main Figures) but the data are still clear. Scale bar = 100 μm. (c) Demonstration that the SA-miR vectors used to drive miR-21 and miR-21∗ achieve elevated expression in human FaDu and HaCaT cells. At least five biological replicates were performed; shown are mean ± s.d. n = 3 independent experiments. ∗P < 0.05,∗∗P < 0.01. N.S. Not Significant. Paired t-tests were used to compare SA-miRs to SA-Scr. (d–f) CRISPR/CAS edited human A431 SCC cells, surveyor assay, and deficiency in tumourigenesis. For both mouse and human SCCs, top three predicted off-target sites were sequenced and no mutations were detected in the three clones tested (see Methods). Shown are mean ± s.d. n = 3 independent experiments. Two-way ANOVA with repeated measurement was performed.

Supplementary Figure 7 More miR-21∗ and miR-21 putative targets in SCCs.

(a) 8 of miR-21∗ and 6 of miR-21 predicted targets are down-regulated in HNSCCs compared to normal tissues. Shown are mean ± s.d. n = 31 for normal tissues and n = 263 for tumours. Unpaired t-test was used to compare normal to tumour samples. (b) Predicted targets that inversely correlate with miR-21∗ or miR-21 levels in tumours, respectively. Shown are mean ± s.d. n = 26 for tumour samples. Paired t-test was used to compare low to high expression tumour samples. (c) In addition to miR-21∗ target PHACTR4 (see main text), RGMA (miR-21∗ target), PDCD4(miR-21 target) and YOD1(miR-21 target) expression levels inversely correlated with poor prognosis in HNSCCs n = 7 ∼ 330 tumour samples. Log-rank test method was used to compare patient survival between high and low expression samples.

Supplementary Figure 8 miR-21∗ targets Phactr4 in SCCs.

(a,b) Western blot showing suppressed levels of Phactr4 resulting from SA-miR-21∗ overexpression (a), and Phactr4 levels from WT or ΔPP1 mutant expression (b). In a, the numbers indicate densitometry values normalized against α-tubulin and measured by ImageJ. At least three biological replicates were performed; shown are representative images.

Supplementary Figure 9 Whole Western Blot gel scan.

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Ge, Y., Zhang, L., Nikolova, M. et al. Strand-specific in vivo screen of cancer-associated miRNAs unveils a role for miR-21∗ in SCC progression. Nat Cell Biol 18, 111–121 (2016). https://doi.org/10.1038/ncb3275

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