Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas - PubMed (original) (raw)

Direct in vivo RNAi screen unveils myosin IIa as a tumor suppressor of squamous cell carcinomas

Daniel Schramek et al. Science. 2014.

Abstract

Mining modern genomics for cancer therapies is predicated on weeding out "bystander" alterations (nonconsequential mutations) and identifying "driver" mutations responsible for tumorigenesis and/or metastasis. We used a direct in vivo RNA interference (RNAi) strategy to screen for genes that upon repression predispose mice to squamous cell carcinomas (SCCs). Seven of our top hits-including Myh9, which encodes nonmuscle myosin IIa-have not been linked to tumor development, yet tissue-specific Myh9 RNAi and Myh9 knockout trigger invasive SCC formation on tumor-susceptible backgrounds. In human and mouse keratinocytes, myosin IIa's function is manifested not only in conventional actin-related processes but also in regulating posttranscriptional p53 stabilization. Myosin IIa is diminished in human SCCs with poor survival, which suggests that in vivo RNAi technology might be useful for identifying potent but low-penetrance tumor suppressors.

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Figures

Fig. 1

Fig. 1. Direct in vivo shRNA screen for HNSCC tumor suppressors

(A) Schematic of pooled shRNA screen. (B) Tumor-free survival of mice of the indicated genotype transduced at E9.5 with the shRNA library targeting putative HNSCC genes (n = number per group; P < 0.0001, log-rank test). (C) Representative pie charts showing percent representation of a particular shRNA within an individual tumor compared to surrounding healthy skin. (D) Top-scoring tumor suppressor candidates and corresponding numbers of tumors showing significant enrichment.

Fig. 2

Fig. 2. Functional validation of Myh9 as a bona fide tumor suppressor

(A) Tumor-free survival of mice of the indicated genotype and shRNA transduction (n > 6 for each genotype, P < 0.0001). Insert shows skin lesions (arrows) on 4-month-old Myh9 shRNA–transduced _T_β_RII_-cKO mouse. (B) Myh9 knockdown results in pulmonary metastases in _T_β_RII_-cKO mice. Metastatic lesions are immunoreactive for epithelial keratin 14 and negative for myosin IIa. (C) Tumor-free survival of Myh9/T_β_RII inducible knockout (iKO) as well as Myh9 heterozygous/T_β_RII iKO and control mice (n = 6, P < 0.001, log-rank test).

Fig. 3

Fig. 3. A noncanonical role for myosin IIa in nuclear retention of activated p53

(A) Tumor-free survival of conditional Trp53 mutant mice transduced with the indicated shRNA (n > 6 for each genotype; no significant survival change could be observed). (B) Myh9 knockdown (kd) but not scrambled control shRNA (c) diminishes p53 and targets p21 (CDKN2) levels in response to the DDR inducer doxorubicin. Myosin IIa and GAPDH levels are shown as controls. (C and D) Lack of nuclear p53 in _Myh9_-cKO versus control (Ctrl) littermate skins 6 hours after γ irradiation (5 Gy). (C) Immunofluorescence (boxed regions show DAPI-stained nuclei in blue); (D) immunoblot analysis. Myosin IIa and GAPDH levels are shown as controls. (E) Quantitative polymerase chain reaction of p53 target genes illustrates the effects of Myh9 knockdown on the p53 pathway. (F) p53 immunoblot of lysates from DDR-induced keratinocytes treated with vehicle (V), blebbistatin (B), Rho kinase inhibitor Y27632 (Y), or latrunculin B (L). GAPDH levels are shown as controls. (G) Nuclear p53 is not retained when DDR-induced Myh9 knockdown primary keratinocytes are exposed to blebbistatin (B). Lamin A/C, IκBα, and γH2AX are controls for nuclear fraction, cytoplasmic fraction, and DDR, respectively. Nuclear export inhibitor leptomycin B rescues the ability of _Myh9_-deficient cells to retain p53 in the nucleus.

Fig. 4

Fig. 4. MYH9 is a bona fide tumor suppressor in human HNSCC

(A) p53 induction in primary human keratinocytes treated with the myosin ATPase inhibitor blebbistatin and with the DDR inducer doxorubicin. GAPDH levels are shown as loading control. (B) Myosin IIa quantifications on 362 samples of human healthy skin, skin SCCs, and HNSCCs. A substantial fraction of cases show absent or reduced myosin IIa expression. (C) Decreased MYH9 mRNA expression correlates with shortened survival. Kaplan-Meier analysis compares overall survival of TCGA HNSCC patients stratified according to the lowest (≤5th percentile) MYH9 expression versus the rest (>5th percentile) (n = 166, P = 0.0044, log-rank test).

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References

    1. Leemans CR, Braakhuis BJ, Brakenhoff RH. Nat. Rev. Cancer. 2011;11:9–22. - PubMed
    1. Stransky N, et al. Science. 2011;333:1157–1160. - PMC - PubMed
    1. Agrawal N, et al. Science. 2011;333:1154–1157. - PMC - PubMed
    1. Beronja S, Livshits G, Williams S, Fuchs E. Nat. Med. 2010;16:821–827. - PMC - PubMed
    1. Berton TR, et al. Oncogene. 2003;22:5415–5426. - PubMed

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