Taking Snapshots of Ciliary Signaling

Although the primary cilium critically influences signaling in development and disease, the organelle’s small size and the dynamic nature of signaling events have posed obstacles to dissecting critical mechanisms. Reporting in Developmental Cell, Mick and colleagues (2015) describe cilia-APEX as an approach to provide dynamic “snapshots” of the ciliary proteome

Tumor-targeted SN38 inhibits growth of early stage non-small cell lung cancer (NSCLC) in a KRas/p53 transgenic mouse model

<div><p>Non-small cell lung cancer (NSCLC) is the leading cause of cancer death worldwide, with a 5-year survival of only ~16%. Potential strategies to address NSCLC mortality include improvements in early detection and prevention, and development of new therapies suitable for use in patients with early and late stage diagnoses. Controlling the growth of early stage tumors could yield significant clinical benefits for patients with comorbidities that make them poor candidates for surgery: however, many drugs that limit cancer growth are not useful in the setting of long-term use or in comorbid patients, because of associated toxicities. In this study, we explored the use of a recently described small molecule agent, STA-8666, as a potential agent for controlling early stage tumor growth. STA-8666 uses a cleavable linker to merge a tumor-targeting moiety that binds heat shock protein 90 (HSP90) with the cytotoxic chemical SN38, and has been shown to have high efficacy and low toxicity, associated with efficient tumor targeting, in preclinical studies using patient-derived and other xenograft models for pancreatic, bladder, and small cell lung cancer. Using a genetically engineered model of NSCLC arising from induced mutation of KRas and knockout of Trp53, we continuously dosed mice with STA-8666 from immediately after tumor induction for 15 weeks. STA-8666 significantly slowed the rate of tumor growth, and was well tolerated over this extended dosing period. STA-8666 induced DNA damage and apoptosis, and reduced proliferation and phosphorylation of the proliferation-associated protein ERK1/2, selectively in tumor tissue. In contrast, STA-8666 did not affect tumor features, such as degree of vimentin staining, associated with epithelial-mesenchymal transition (EMT), or downregulate tumor expression of HSP90. These data suggest STA-8666 and other similar targeted compounds may be useful additions to control the growth of early stage NSCLC in patient populations.</p></div

STA-8666 is more effective than irinotecan in controlling the growth of NSCLC cell lines <i>in vitro</i>.

<p><b>A</b>. Quantification of CellTiterBlue (CTB) viability assay in human A549 and H441 NSCLC cell lines treated with vehicle, STA-8666 and irinotecan in doses of 0.1, 1, 10 μM for up to 5 days. Chart curves represent average data from two independent runs performed with duplicate samples. <b>B-E</b>. Western blot analysis of phosphorylated (p) and total (t) ERK1/2 (<b>B</b>), AKT (<b>C</b>), CHK2 (<b>D</b>) and CDK1 (<b>E</b>) protein expression in human A549 and H441 NSCLC cell lines, at 48 hours after treatment with vehicle (V), STA-8666 and irinotecan in doses of 0.1, 1, or 10 μM, as indicated. Histograms represent data from two independent experiments. All graphs: *, p≤0.05; **, p≤0.01; ***, p≤0.001 relative to vehicle controls.</p

STA-8666 treatment prevents tumor growth <i>in vivo</i>.

<p><b>A</b>. Representative H&E stained lungs. Arrows indicate tumors. Magnification: 4x. Scale bars: 2mm, top panels; 850 μm, bottom panels. <b>B.-D</b>. Quantification of total tumor surface area (<b>B</b>), number of independent tumors (<b>C</b>), and average size of individual tumors (<b>D</b>).</p

Statistical analysis of correlations in between markers expression in vehicle and STA-8666 treated mice cohorts.

<p><b>A-D</b>. Shown Spearman nonparametric correlation coefficient with two-tailed p-value between H-score for Ki-67 and tumor area (<b>A</b>), H-score for cleaved caspase 3 and tumor area (<b>B</b>), H-scores for Ki-67 and cleaved caspase 3 (<b>C</b>), and H-scores for Ki-67 and HSP90 (<b>D</b>). Each dot represents an individual mouse. P values shown in red are for STA-8666 treatment cohort, for blue are for vehicle treatment cohort.</p

<i>In vitro</i> benchmarking of STA-8666 and irinotecan for EMT-related markers.

<p><b>A, B</b>. Western blot analysis of expression of E-cadherin, vimentin, and Snail in human A549 (<b>A</b>) and H441 (<b>B</b>) NSCLC cell lines 48 hours after treatment with vehicle (V), STA-8666 or irinotecan at the doses of 0.1, 1, 10 μM, as indicated. Histograms represent data from two independent experiments, normalized to vehicle (V) controls.</p

Adeno-cre induced murine lung cancer model.

<p><b>A</b>. Experimental design. <b>B</b>. Representative MRI images of murine lungs and tumors (arrows) from the vehicle and STA-8666 treatment cohorts. <b>C</b>. Quantified tumor volume determined by MRI in vehicle versus STA-8666 treated mice. <b>D</b>. Body weight dynamics in mice receiving vehicle or STA-8666.</p

Signaling consequences of STA-8666 treatment.

<p><b>A.-F</b>. Left, Representative images and right, quantification of IHC or immunofluorescence for Ki-67 (<b>A</b>), cleaved caspase 3 (<b>B</b>), pS⁸²⁴-KAP1 (<b>C</b>), pT²⁰²/Y²⁰⁴-ERK1/2 (<b>D</b>), vimentin (light red) and DAPI (blue) (<b>E</b>) and HSP90 (<b>F</b>). Magnification: 20x. Scale bars: 75 μm, except for vimentin, where scale bar is 40 μm.</p