Silencing of CDK2, but not CDK1, separates mitogenic from anti-apoptotic signaling, sensitizing p53 defective cells for synthetic lethality

<p>Small molecule inhibitors targeting CDK1/CDK2 have been clinically proven effective against a variety of tumors, albeit at the cost of profound off target toxicities. To separate potential therapeutic from toxic effects, we selectively knocked down CDK1 or CDK2 in p53 mutated HACAT cells by siRNA silencing. Using dynamic, cell cycle wide proteome arrays, we observed minor changes in overall abundance of proteins critically involved in cell cycle transition despite profound G₂/M or G₁/S arrest, respectively. Employing phospho site specific analyses, we identified uncoupled mitogenic, yet pro-apoptotic signaling from counter balancing anti-apoptotic activity in CDK2 disrupted cells. Moreover, a crucial role of CDK2 activity in early serum response was observed, extending well-established roles of CDKs outside their cell cycle regulating functions. In contrast, disruption of CDK1 only marginally affected phosphorylation events of crucial signaling nodes prior to G₂/S transition. The data presented here suggest that the temporal separation of pro- and anti-apoptotic pathways by selective inhibition of CDK2 disrupts coherent signaling modules and may synergize with anti-proliferative drugs, averting toxic side effects from CDK1 inhibition.</p

<i>HMGA1</i> is a direct target of <i>let-7b</i> in prostate cancer.

<p>A. 3′UTR-sequence of binding sites for <i>let-7b</i> are indicated by vertical lines towards <i>HMGA1</i>. B. qRT-PCR for <i>let-7b</i> and <i>HMGA1</i> in untreated LNCaP and PC-3 cells. While <i>let-7b</i> shows higher expression in LNCaP compared to PC-3 cells, <i>HMGA1</i> expression shows inverse expression to <i>let-7b</i>. C. qRT-PCR and Western Blot analysis for <i>HMGA1</i> using lysates from PC-3 cells transiently transfected with pre−/anti-<i>7b</i> and negative control, respectively. Western Blot analysis was performed in two independent experiments. Band intensity was measured by Image J in regard to housekeeping gene (<i>β-Actin</i>) band intensity. D. <i>HMGA1</i> as target of <i>let-7b</i>. Luciferase construct containing the 3′UTR of <i>HMGA1</i> were transfected with either pre–<i>let-7b</i> or pre-miR control into PC-3 cells. Relative expression of firefly luciferase was determined 48 h post transfection and normalized to the transfection control. Error bars represent +/− standard deviation and were calculated using three independent experiments. E. Correlation analysis of <i>HMGA1</i> and <i>let-7b</i> in vivo. In 21 pairs of PCa and adjacent benign tissue qRT-PCR analysis for <i>let-7b</i> and <i>HMGA1</i> were performed. Expression levels, normalized by <i>RNU6B</i> or <i>β-Actin</i>, were used to calculate the Pearson’s correlation coefficient (r = -0.71). B. + C. Relative expression is shown by means of 3 independent qRT-PCR experiments. *indicate statistically significant results (p<0.05). P values were calculated using the Welch 2 sample t-test.</p

Microarray analysis in high-risk PCa and BPH tissue.

<p>A. Microarray profiling of 6 BPH and 13 high-risk PCa tissue specimens (cohort A). The latter were subdivided by clinical outcome: group 1: high-risk PCa+CPFS (n = 7); group 2: high-risk PCa+CF (n = 6). Row-wise scaled heatplot of genes showing a fold change >1.5 and an adjusted p value <0.05. Red indicating high expression, green low expression. Cancerous and non-cancerous prostate tissues are clearly separated. Clustering indicates a distinction between group 1 and 2. B. Technical validation of the microarray data by qRT-PCR. Relative expression of <i>miR-16</i>, <i>-29a</i>, and <i>-221</i> was analyzed in 6 BPH (white) and 13 high-risk PCa samples (grey). MiRNA expression is shown as means of BPH and high-risk PCa expression with error bars for standard deviation. *indicates p<0.01, p values were calculated using the Welch 2 sample t-test.</p

MiRNA expression in high-risk PCa (cohort A) with early clinical failure.

<p>A. Venn diagram showing relationships between human miRs that were expressed significantly different (±1.5 fold) in PCa group 1 vs. PCa group 2 vs. BPH (adj p<0.05). Circles include numbers of up- or down-regulated human miRs of each pair-wise comparison. Common miRs between different comparisons are shown in intersections. Grey boxes indicate expression status of the <i>let-7</i> family members. The table below lists all human miRs in which expression was significantly different either: between (gr.1 vs. gr.2), (gr.2 vs. BPH) and (gr.1 vs. BPH) (red column); between (gr. 1 vs. gr. 2) and (gr. 2 vs. BPH) (green column); or between (gr. 1 vs. gr. 2) and (gr. 1 vs. BPH) (blue column). All miRNAs are ranked based on the maximum expression fold change. The two columns aside the list of miRNAs indicate an up- or down-regulation in expression (see arrows) and the dimension of expression fold change. B. Validation of the microarray data by qRT-PCR. Relative expression of <i>miR-146b</i>, <i>-181b</i>, and <i>let-7a</i>/<i>b</i>/and <i>-c</i> was analyzed in 6 BPH (white), 7 high-risk PCa samples with CPFS (group 1) (light grey), and 6 high-risk PCa samples with CF (group 2) (dark grey). MiRNA expression is shown as means of expression in BPH and high-risk tissue, respectively with error bars for standard deviation. *indicates p<0.01, p values were calculated using the Welch 2 sample t-test.</p

Uni- and multivariate Cox regression analysis of high-risk cohort B

<p>Uni- and multivariate Cox regression analysis of high-risk cohort B</p

Clinico-pathological features of both high-risk PCa groups (cohort A – test group and cohort B – validation group).

<p>Clinico-pathological features of both high-risk PCa groups (cohort A – test group and cohort B – validation group).</p

Kaplan-Meier analysis for BCR and CF in a learning and testing high-risk PCa cohort.

<p>Kaplan-Meier analysis for biochemical and clinical relapse of 98 and 92 patients with high-risk disease (cohort A and B). Groups were dichotomized in low and high <i>let-7b</i> and -<i>7c</i> expression. Survival curves were generated using the Bioconductor package “survival”. A. Kaplan Meier analysis of cohort A, the learning data set (n = 98). B. Kaplan Meier analysis of cohort B, the testing data set (n = 92). Low <i>let-7b</i> expression is associated with BCR and CF.</p

IL-10 producing Tregs are apoptosis prone.

<p>(<b>A</b>) CD4⁺CD25⁺ Tregs from CD28SA treated DEREG mice were isolated and stimulated over night with anti-CD3, anti-CD28, IL-2. Next day cells were stained for IL-10 and separated into IL-10⁺Foxp3⁺ (black line) and IL-10⁻Foxp3⁺ (grey line) via FACS Sorter and cultured for 1 to 3 days with anti-CD3, anti-CD28, IL-2 and examined for Annexin V and 7AAD positive cells. As control unseparated CD4⁺CD25⁺ T-cells (dashed line) were used. Graphs show means ± SD from 3 individual experiments. ** p<0.005 compared IL-10⁺Foxp3⁺ to IL-10⁻Foxp3⁺ cells. (<b>B</b>) Flow cytometric analysis of costaining of Annexin V and clone 10-7A (phospholipids). (<b>C</b>) Binding of clone 10-7A given in ΔMFI ( = MFI(marker)−MFI(isotype)) on gated CD4⁺ T-cells 3 days after CD28SA injection dependent on IL-10 and Foxp3 expression. Graphs show means ± SD from 3 or 5 mice assayed individually and results are representative of two independent experiments. ** p<0.005. (<b>D</b>) Gene expression data given in M (ln(fold change)) of functional cluster “apoptosis” (DAVID) of sorted IL-10⁺ and IL-10⁻ CD4⁺CD25⁺ Tregs 3 days after stimulation. Transcripts selected for genes that showed ≥2-fold change and an adjusted p-value<0.001 (n = 2). (<b>E</b>) Bcl-2 expression of gated CD4⁺ T-cells 3 days after CD28SA injection dependent on IL-10 and Foxp3 expression. * p<0.05. Results are representative of three independent experiments with 3–4 mice.</p

Selected AS (<i>TRIM47</i>, <i>WRAP53</i>, <i>CHN1</i>; left) and RG (<i>DTL</i>, <i>SLFN5</i>; right) gene views showing probe set intensity plots (graphs) for each stimulated only (S, blue) or stimulated/inhibited (I, red) samples.

<p>Exons assigned with probe sets, location of primers within constant and AS regions and expected fragment lengths on RT-PCR analyses (which are exemplified each right to the corresponding graphs; one out of at least three independent experiments is shown) are indicated below the transcript profiles (A). Samples prepared as in A. were used for qPCR analyses with amplification levels for S (white bars) and I (black bars) being indicated. Data shown were obtained using a selected pair of RNAs (S and I), assays were performed in triplicate (B).</p

ABA induces methylation and reduces transcript levels.

<p>(A) The difference in the levels of transcription (Transcription logFC) and DNA methylation (Methylation logFC) were determined from samples treated with ABA versus samples not treated with ABA for the <i>A. thaliana</i> genes <i>NDF4</i> (At3g16250), <i>SIG5</i> (At5g24120) and <i>F12A4.4</i> (At1g35420). Transcript levels were determined by qRT-PCR and methylation profiles of upstream regions by bisulfite sequencing. For these experiments <i>A. thaliana</i> seedlings were treated with ABA for two days according to the protocol of Nishimura <i>et al</i>. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003267#pgen.1003267-Nishimura1" target="_blank">[34]</a>. (B) Changes in methylation and transcription in the crown gall tumor compared to mock-iocculated stems according to methylome (Methylation logFC) and transcriptome data (Transcription logFC) of a previous study <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003267#pgen.1003267-Deeken1" target="_blank">[8]</a>. Each data set is based on at least three independent biological replicates and the logarithmic of fold changes (logFC) were calculated from the mean values.</p