Nek4 status differentially alters sensitivity to distinct microtubule poisons

Abstract

Microtubule poisons are widely used in cancer treatment, but the factors determining the relative efficacy of different drugs in this class remain obscure. In this study, we identified the NIMA kinase Nek4 in a genetic screen for mediators of the response to Taxol, a chemotherapeutic agent that stabilizes microtubules. After Taxol treatment, Nek4 promoted microtubule outgrowth, whereas Nek4 deficiency impaired G(2)-M arrest and decreased formation of mitotic-like asters. In contrast, Nek4 deficiency sensitized cells to vincristine, which destabilizes microtubules. Therefore, Nek4 deficiency may either antagonize or agonize the effects of microtubule poisons, depending on how they affect microtubule polymerization. Of note, Nek4 gene maps to a commonly deleted locus in non-small cell lung cancer. Thus, Nek4 deletion in this disease may rationalize the use of particular types of microtubule poisons for lung cancer therapy.

Figure 1. RNAi screening identifies Nek4 as a regulator of microtubule poison-induced cell death

(A)In vitro screening methodology. Lymphoma cells were partially infected with 48 pools of 48 distinct shRNAs, treated with taxol (4, 6, and 8nM) and monitored using GFP-based flow cytometry for changes in the relative percentage of shRNA-containing (GFP+) cells. Genomic DNA from enriched pools was subsequently subjected to shRNA-specific PCR and sequenced to determine relative shRNA abundance. (B) An in vitro GFP competition assay comparing relative taxol sensitivity in cells infected with two distinct shRNAs targeting Nek4 (6nM taxol, 48h post-treatment; n=5 for all samples). (C) qRT-PCR (n≥3) and western blot analysis of Nek4 expression in lymphoma cells. (D) Partially-transduced lymphoma cells were separately treated with doxorubicin (10ng/ml), cisplatin (7.5ng/ml), 5-fluorouracil (40ng/ml) and vincristine (1.5nM) at similar levels of cytotoxicity (~90% cell death at 48h). The percentage of GFP+ cells was determined 48 hours post treatment (n=3 for doxorubicin, 5-FU, cisplatin, and n=5 for vincristine treatments). Values are shown with standard deviations (s.d.). P-values were determined using a Student’s t-test.

Figure 2. Nek4 knockdown promotes resistance to taxol, while sensitizing cells to vincristine

(A) (Above) A western blot showing Nek4 knockdown in lung adenocarcinoma cells expressing shNek4-2. (Below) shNek4-2 transduced cells were treated with 5μM taxol or vincristine and monitored for cell survival relative to cells expressing a vector control (n=3 independently treated samples for each drug, +/− s.d.). (B) G2/M arrest profiles in lung adenocarcinoma cells expressing shNek4-2. Knockdown cells were treated with 5μM taxol or 5μM vincristine for 8 hours and analyzed for DNA content by flow cytometry to determine the extent of the microtubule poison-induced G2/M arrest. Shown is the 4N/2N ratio (indicative of an arrest) normalized to matched vector control cells (n=3 for all samples +/− s.d.). P-values were determined using a Student’s t-test.

Figure 3. Nek4 knockdown cells show altered microtubule phenotypes

(A) LA cells expressing a Nek4 shRNA or a control vector were treated with 5μM taxol for 4h and then stained with anti-α-tubulin to visualize microtubules. Microtubule asters (red arrowheads) and bundles (yellow arrowheads) were observed under these conditions. (B) (Left graph) Quantification of aster containing cells in shNek4 and vector control infected cell populations using OpenLab5 software (n=3 independently treated replicates, 6 averaged fields/sample). (Right) Phospho-histone H3 staining of drug treated LA cells failed to show any significant change in the mitotic index in the presence or absence of Nek4 (n=3 for all samples +/− s.d.). (C) In a microtubule repolymerization assay, lung adenocarcinoma cells were allowed to repolymerize microtubules following transient (0.5μM, 30 minutes) nocodazole treatment. Qualitative differences were apparent at one (third column) and two (fourth column) minutes post-nocodazole release. (D) Quantification of the repolymerization defect in shNek4-2 LA cells. Microtubule lengths are shown at the one and two-minute time points, respectively. The average microtubule length in each sample is indicated with a red bar. P-values were determined using a Student’s t-test.

Figure 4. Nek4 suppression alters the response to microtubule poisons in vivo

(A) Schematic depicting in vivo experimental approaches. Partially transduced (upper row) or GFP-sorted (lower row) lymphoma cells were injected into recipient mice and allowed to develop into palpable lymphomas. Resulting tumors were then treated with taxol or vincristine and then either harvested to examine the percentage of GFP+ cells or monitored for tumor-free and overall survival rates. (B) Partially transduced shNek4-2 lymphomas were harvested 24 hours post drug treatment (25mg/kg taxol or 1.0mg/kg vincristine) and analyzed by flow cytometry for changes in GFP percentage. Taxol treatment resulted in an increase in the percentage of GFP+ cells (compared to pre-injection GFP levels), while vincristine-treated tumors displayed dramatic selection against Nek4 knockdown. P-values were determined using a Student’s t-test (C) Kaplan-Meier survival curve depicting overall survival of shNek4-2 or control tumor bearing mice following treatment with a maximally tolerated dose (1.5mg/kg) of vincristine (vector, n=13; shNek4-2, n=12). P-values were determined using a log rank test.

Figure 5. Nek4-dependent differential sensitivity to microtubule poisons in human lung adenocarinoma cells

(A) Four human lung adenocarcinoma cell lines were separately treated with taxol and vincristine and examined for viability 48 hours after treatment. Viability comparisons (left, graph) at a fixed drug dose of 5μM taxol or vincristine revealed one cell line (colo669) with a significantly lower taxol/vincristine survival ratio. This cell line also had high baseline levels of Nek4 protein (right, western blot). (B) Colo669 cells were retrovirally infected with shRNAs targeting human Nek4 and subjected to in vitro survival assays. Knockdown cells with significant depletion of Nek4 protein (right, western blot) also demonstrated relative resistance to taxol and sensitivity to vincristine (left, shown as an upward shift in the taxol/vincristine survival ratio). (C) Transduction of ‘low-Nek4’ sklu1 cells with Nek4 shRNAs did not affect their relative sensitivity to taxol and vincristine. (below) Western blot showing Nek4 levels following transduction of sklu1 cells with Nek4 shRNAs or a Nek4 cDNA. (D) Sklu1 cells stably overexpressing full length Nek4 cDNA were treated with either taxotere or vincristine for 48h, at which time cell viability was assessed using CellTiterGlo reagents. Mann-Whitney statistical comparison of EC50 values derived from best-fit non-linear regression curves revealed increased sensitivity (avg. EC50 for vector control=10.5μM and NEK4=5.5μM, p=0.002) to taxotere and resistance to vincristine (avg. EC50 for vector control=3.3μM and NEK4=6.4μM, p=0.002) in cells overexpressing Nek4. Data was generated from three independent experiments for each drug.