Journal: The Journal of Clinical Investigation

Article Title: A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma

doi: 10.1172/JCI43897

Figure Lengend Snippet: (A) Knockdown of endogenous p63 RNA (top) and protein (bottom) by p63-directed or control (Ct) lentiviral shRNA in JHU-029 human SCC cells at 48 hours, in duplicate experiments (Exp). β-Tubulin (β-Tub) loading control. (B) Array analysis showing the fold change and direction of change for all miRs regulated at 1.5-fold or more in p63-ablated versus control samples shown in A. Circles show miRs predicted to target p73. (C) Validation of p63 repression of miRs targeting p73 by real-time quantitative RT-PCR (QRT-PCR) at 72 hours after lentiviral p63-directed or control shRNA expression in JHU-029 cells. (D) The p63-regulated miRs repress gene expression via the p73 3′ UTR. Cotransfection of the indicated miR mimics or control (scrambled) miR, together with the UTR-reporter or control reporter; results show relative luciferase units (RLU) normalized to the control miR. Note that repression correlates with the number of predicted seed-binding sequences (Sites) for each miR. (E) A miR-dependent mechanism for regulation of the p73 3′ UTR by p63. Lentiviral shRNA knockdown of Drosha followed by cotransfection of the UTR or control reporter, together with either a p63 shRNA or ΔNp63α cDNA or their respective controls in JHU-029 cells. RLU values expressed as p63 knockdown/control or ΔNp63α overexpression/control (OV/Ct). Above, immunoblot shows efficient Drosha knockdown. All error bars show SEM for triplicate measurements from representative experiments.

Article Snippet: Taken together, these data show that p63 is an endogenous repressor of this miR in normal human and murine cells and in the context of tumor-specific p63 overexpression. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Endogenous p63 and p73 mediate miR-193a regulation by chemotherapy. ( A ) Regulation of human miR-193a-5p (hereafter miR-193a) and murine miR-193* by endogenous p63, measured by QRT-PCR 48 hours after lentiviral p63 knockdown in JHU-029 cells (029), murine SCC cells (mSCC), human immortal primary keratinocytes (OKF6), and control A549 cells, which do not express p63. ( B ) Repression of endogenous miR-193a by p63 requires DNA binding.

Techniques: Knockdown, Control, shRNA, Biomarker Discovery, Quantitative RT-PCR, Expressing, Gene Expression, Cotransfection, Luciferase, Binding Assay, Over Expression, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma

doi: 10.1172/JCI43897

Figure Lengend Snippet: (A) Regulation of human miR-193a-5p (hereafter miR-193a) and murine miR-193* by endogenous p63, measured by QRT-PCR 48 hours after lentiviral p63 knockdown in JHU-029 cells (029), murine SCC cells (mSCC), human immortal primary keratinocytes (OKF6), and control A549 cells, which do not express p63. (B) Repression of endogenous miR-193a by p63 requires DNA binding. Retroviral expression of WT or non–DNA-binding ΔNp63α mutant (R304W) was followed by QRT-PCR. (C) Regulation of murine miR-193* by endogenous p63 in vivo. Homozygous p63flox mice with or without expression of the K14-Cre/ER transgene (Tg) were treated with tamoxifen (Tam) or vehicle control for 5 days, and epidermal cells were collected 21 days later for RNA analysis by QRT-PCR. (D) Inverse correlation between p63 expression and miR-193a expression in human primary HNSCC specimens, assessed by QRT-PCR. df, degrees of freedom. (E) Rapid upregulation of endogenous PUMA (top) and miR-193a (bottom) following tetracycline-induced (Tet) TAp73 expression in JHU-029 cells. Middle panels show induced TAp73 expression by immunoblot. (F) Endogenous p73-dependent induction of miR-193* in murine SCC cells by cisplatin. Following preinfection with lentiviral TAp73 shRNA or control, cells were treated with cisplatin (Cis, 4 μM, 24 hours) or vehicle control (Veh) prior to RNA analysis. (G) Regulation of miR-193* is independent of p53. WT or p53–/– E1A-immortalized MEFs were treated with doxorubicin (Dox) (0.2 μM, 12 hours) or vehicle control prior to RNA analysis. Bax and Noxa are induced in a p53-dependent manner, whereas miR-193* induction correlates only with TAp73. All error bars show SEM for triplicate measurements.

Article Snippet: Taken together, these data show that p63 is an endogenous repressor of this miR in normal human and murine cells and in the context of tumor-specific p63 overexpression. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Endogenous p63 and p73 mediate miR-193a regulation by chemotherapy. ( A ) Regulation of human miR-193a-5p (hereafter miR-193a) and murine miR-193* by endogenous p63, measured by QRT-PCR 48 hours after lentiviral p63 knockdown in JHU-029 cells (029), murine SCC cells (mSCC), human immortal primary keratinocytes (OKF6), and control A549 cells, which do not express p63. ( B ) Repression of endogenous miR-193a by p63 requires DNA binding.

Techniques: Quantitative RT-PCR, Knockdown, Control, Binding Assay, Retroviral, Expressing, Mutagenesis, In Vivo, Western Blot, shRNA

Journal: The Journal of Clinical Investigation

Article Title: A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma

doi: 10.1172/JCI43897

Figure Lengend Snippet: (A) p73-dependent inhibition of viable cells following miR-193* antagomir (anti-miR) treatment. Murine SCC (mSCC) cells were infected with lentiviral p73 shRNA or control, then treated with a miR-193* antagomir or scrambled control followed by cell counts at 72 hours. (B) miR-193a abrogates cisplatin-induced growth inhibition. JHU-029 cells were pretreated with miR-193a mimic or scrambled control (miR Ct) for 24 hours followed by 1 hour cisplatin treatment, and cell counts were determined by XTT assay 72 hours later. (C) Enhancement of cisplatin sensitivity by the miR-193a antagomir is p73-dependent. JHU-029 cells were infected with lentiviral TAp73 shRNA or control, then treated for 24 hours with a miR-193a antagomir or scrambled control (anti–miR Ct) prior to 1 hour cisplatin treatment and cell counts 72 hours later. (D) Endogenous miR-193a controls cisplatin-mediated clonogenic suppression. JHU-029 cells were treated for 24 hours with a miR-193a antagomir (anti–miR-193a) or scrambled control (anti–miR Ct) prior to cisplatin treatment (0.5 μm, 1 hour), then plated at clonal density for colony counts. Scale bar: 1 cm. *34.7% repression; **64.5% repression. (E) Control of p73 activity by endogenous miR-193a. Cells treated as in D were harvested at 24 hours after cisplatin for analysis of the p73 target gene NOXA by QRT-PCR. Error bars show SEM.

Article Snippet: Taken together, these data show that p63 is an endogenous repressor of this miR in normal human and murine cells and in the context of tumor-specific p63 overexpression. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Endogenous p63 and p73 mediate miR-193a regulation by chemotherapy. ( A ) Regulation of human miR-193a-5p (hereafter miR-193a) and murine miR-193* by endogenous p63, measured by QRT-PCR 48 hours after lentiviral p63 knockdown in JHU-029 cells (029), murine SCC cells (mSCC), human immortal primary keratinocytes (OKF6), and control A549 cells, which do not express p63. ( B ) Repression of endogenous miR-193a by p63 requires DNA binding.

Techniques: Inhibition, Infection, shRNA, Control, XTT Assay, Activity Assay, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma

doi: 10.1172/JCI43897

Figure Lengend Snippet: (A) Inverse correlation between miR-193a and p73 expression in primary human HNSCC specimens, assessed by QRT-PCR. (B) Murine SCCs resemble human SCC, assessed by histology (H&E) and p63 immunohistochemical staining. Original magnification, ×200. (C) Anti–miR-193* inhibits tumor growth in the absence of cisplatin, and it enhances cisplatin sensitivity. Primary disaggregated SCC was treated with miR-193* antagomir or scrambled control, then implanted into nude mice, followed by cisplatin treatment (5 mg/kg) or vehicle. n = 10 per arm. *P < 0.05 by 2-way repeated measures ANOVA. (D) Increased expression of activated caspase-3 (brown staining) in vivo following antagomir-mediated miR-193* inhibition compared with control antagomir treatment, both in the presence and absence of cisplatin treatment. Original magnification, ×200. The percentage of cleaved caspase-3–positive cells ± SEM is shown below each panel, based on 100 cells counted in 3 representative fields. (E) Representative mice following cisplatin treatment.

Article Snippet: Taken together, these data show that p63 is an endogenous repressor of this miR in normal human and murine cells and in the context of tumor-specific p63 overexpression. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Endogenous p63 and p73 mediate miR-193a regulation by chemotherapy. ( A ) Regulation of human miR-193a-5p (hereafter miR-193a) and murine miR-193* by endogenous p63, measured by QRT-PCR 48 hours after lentiviral p63 knockdown in JHU-029 cells (029), murine SCC cells (mSCC), human immortal primary keratinocytes (OKF6), and control A549 cells, which do not express p63. ( B ) Repression of endogenous miR-193a by p63 requires DNA binding.

Techniques: Expressing, Quantitative RT-PCR, Immunohistochemical staining, Staining, Control, In Vivo, Inhibition

Journal: The Journal of Clinical Investigation

Article Title: A microRNA-dependent program controls p53-independent survival and chemosensitivity in human and murine squamous cell carcinoma

doi: 10.1172/JCI43897

Figure Lengend Snippet: Top: in SCC, p63 is an inhibitor of p73 and a transcriptional repressor of miRs that target p73. The miR-193a/193* is a direct transcriptional target of both p63 and p73 and a direct feedback inhibitor of p73. Middle: cisplatin treatment induces p63 degradation and p73 activation, thereby inducing this miR, which then limits p73-dependent chemosensitivity through direct feedback inhibition. Bottom: this feedback loop is disrupted by miR inhibition, which increases p73 levels and enhances chemosensitivity.

Article Snippet: Taken together, these data show that p63 is an endogenous repressor of this miR in normal human and murine cells and in the context of tumor-specific p63 overexpression. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 Endogenous p63 and p73 mediate miR-193a regulation by chemotherapy. ( A ) Regulation of human miR-193a-5p (hereafter miR-193a) and murine miR-193* by endogenous p63, measured by QRT-PCR 48 hours after lentiviral p63 knockdown in JHU-029 cells (029), murine SCC cells (mSCC), human immortal primary keratinocytes (OKF6), and control A549 cells, which do not express p63. ( B ) Repression of endogenous miR-193a by p63 requires DNA binding.

Techniques: Activation Assay, Inhibition