Mechanisms promoting escape from mitotic stress-induced tumor cell death

Abstract

Non-small cell lung cancer (NSCLC) is notorious for its paltry responses to first-line therapeutic regimens. In contrast to acquired chemoresistance, little is known about the molecular underpinnings of the intrinsic resistance of chemo-naïve NSCLC. Here we report that intrinsic resistance to paclitaxel in NSCLC occurs at a cell-autonomous level because of the uncoupling of mitotic defects from apoptosis. To identify components that permit escape from mitotic stress-induced death, we used a genome-wide RNAi-based strategy, which combines a high-throughput toxicity screen with a live-cell imaging platform to measure mitotic fate. This strategy revealed that prolonging mitotic arrest with a small molecule inhibitor of the APC/cyclosome could sensitize otherwise paclitaxel-resistant NSCLC. We also defined novel roles for CASC1 and TRIM69 in supporting resistance to spindle poisons. CASC1, which is frequently co-amplified with KRAS in lung tumors, is essential for microtubule polymerization and satisfaction of the spindle assembly checkpoint. TRIM69, which associates with spindle poles and promotes centrosomal clustering, is essential for formation of a bipolar spindle. Notably, RNAi-mediated attenuation of CASC1 or TRIM69 was sufficient to inhibit tumor growth in vivo. On the basis of our results, we hypothesize that tumor evolution selects for a permissive mitotic checkpoint, which may promote survival despite chromosome segregation errors. Attacking this adaptation may restore the apoptotic consequences of mitotic damage to permit the therapeutic eradication of drug-resistant cancer cells.

Figure 1

(A) Paclitaxel dose curves in indicated NSCLC cell lines. Each point is the mean ± Standard Error of the Mean (SEM) for 3 independent experiments. (B) Whole cell lysates (WCLs) from HCC366 and H1155 exposed to paclitaxel for 48 hours were immunoblotted as indicated. (C) Immunostaining of HCC366 cells treated for 24 hours. Arrowheads indicate micronucleated cells. Scale = 5 μm or 15 μm (right panel). (D) Single-cell lineage tracing of HCC366 cells stably expressing GFP-H2B (HCC366-GFP-H2B) for 48 hours post-paclitaxel exposure. Each circle represents a single cell. Bar is mean mitotic transit time, which is also indicated. Right panel: Cells were transfected with indicated siRNAs for 48 hours prior to drug exposure. (E) Quantitation of single-cell lineage tracing of HCC366-GFP-H2B micronucleates post exposure to 10 nM paclitaxel. Bars represent mean ± range for 100 cells over 2 experiments. (F) H&E staining of HCC366 subcutaneous tumor xenografts treated as indicated. Arrowheads specify micronucleated cells. Scale = 40 μm. Zoomed images are of white boxed area.

Figure 2

(A) Graph of relative mitotic transit time (y) vs. relative caspase-3/7 activity (APO-ONE®) in HCC366 cells exposed to paclitaxel. A subset of siRNA gene targets is indicated. (B) Colony formation assay in HCC366s exposed to 10 nM paclitaxel for 48 hours. Bars represent mean ± SEM from n=3 independent experiments. (C) HCC366-GFP-H2B cells were transfected with indicated siRNAs for 48 hours followed by exposure to 10 nM paclitaxel. Live cell imaging commenced with paclitaxel treatment and single-cell lineage traces of 50 cells from 2 independent experiments were measured. (D) As in C, but in the presence of siMAD2. (E) WCLs from HCC366 cells transfected for 48 hours followed by exposure to vehicle or 10 nM paclitaxel for 48 hours were immunoblotted with indicated antibodies. (F) WCLs from non-tumorigenic HBEC3KT (N) and HCC366 (T) cells treated as in 2E were immunoblotted with indicated antibodies. (G) Left panel: Relative cleaved caspase-3/7 activity (APO-ONE®) in HCC366s treated with vehicle, 10 nM paclitaxel or 2.5 μM proTAME for 24 hours. Bars represent mean ± SEM for n=9 independent experiments. ** P <0.01 *** P < 0.0001 (unpaired two-tailed student's t-test). Right panel: Immunoblot of WCLs of HCC366s treated as in left panel.

Figure 3

(A) WCLs of NSCLC cells immunoblotted with indicated antibodies following siRNA transfection for 48 hours and subsequent treatment as indicated for 48 hours. siPLK1 was used as a positive control for cleaved caspase-3 detection in HBEC3KT cells. (B) As in A, except paclitaxel treatment was 24 hours prior to immunostaining. Scale = 5 μm. (C) Top panel: H1299 cells were transfected for 72 hours with indicated siRNAs followed by glutaraldehyde fix and immunostained. Scale =15 μm. Bottom panel: Immunoblots of H1299 cells following an in vivo polymerized tubulin assay (D) In vivo polymerized tubulin assay in H1299 cells transfected for 72 hours. Lysates were immunoblotted with indicated antibodies. (E) HCC366 cells treated as in B, were fixed in cold methanol and immunostained as indicated. Scale = 5 μm. BUBR1 foci were counted by manual inspection in 60 cells per experiment in 3 independent experiments. Bar indicates mean. *** P < 0.001, Mann-Whitney t-test.

Figure 4

(A&B) As in 3A&B. Scale bars = 10 μm for A549 and H1299, 5 μm for HCC366. (C) As in 3E. ** P <0.05.

Figure 5

(A) HeLa cells transfected with indicated cDNAs for 48 hours were fixed and immunostained. Scale = 5μm. Right panel: Quantitation of cells from (A). Bars represent mean ± range for n=2 independent experiments. (B) Immunostaining of H1299-myc-TRIM69A representing each stage in the cell cycle based on nuclear morphology. Scale bar = 5 μm. (C) H1299 cells stably expressing myc-TRIM69A were transfected with indicated siRNA for 72 hours and immunostained with indicated antibodies. Plots represent relative fluorescence of myc-TRIM69A at centrosomes in > 150 cells across 3 independent experiments. *** P <0.0001 by two-tailed Mann Whitney test. Scale = 5 μm. (D) Left panel: H1299 cells were immunostained with indicated antibodies following siRNA transfection for 72 hours. Scale = 5 μm. Middle panel: Quantitation of cells from (D). Bars represent mean ± range from 2 independent experiments. Right panel: Bars represent mean mRNA expression levels of HAUS1 72 hours post-transfection.

Figure 6

(A) Bars represent mean mRNA expression levels ± range of CASC1 and TRIM69 in HCC366 cells expressing indicated hairpins from 2 independent infections. (B) Tumor growth curves for mice harboring HCC366s expressing indicated shRNAs. shGFP n=9, shCASC1 n=9 and shTRIM69 n=7. Mean ± standard deviation (SD). (C) Tumors expressing indicated short hairpins were excised 96 days following injection. Left panel: Bars represent mean mass of tumors ± SEM; shGFP n=7, shCASC1 n=10, shTRIM69 n=8. Right: Representative images of excised tumors. (D) Tumors from (B) were sectioned and H&E stained. Left: Quantitation of micronucleated cells in tumor sections. 6 independent tumors were examined for each indicated shRNA. Right: Representative images of H&E stained tumor sections. Scale = 50 μm. White arrowheads indicate micronucleated cells. Blue arrowheads indicate aberrant mitotic cells. (E) Tumors from (B) were sectioned and TUNEL stained. Bars indicate mean TUNEL positive cells ± SEM from 6 tumors for each shRNA condition. For all panels, ** P <0.01. *** P < 0.0001 (unpaired two-tailed student's t-test).