Integrative mRNA profiling comparing cultured primary cells with clinical samples reveals PLK1 and C20orf20 as therapeutic targets in cutaneous squamous cell carcinoma

Abstract

Identifying therapeutic targets for cancer treatment relies on consistent changes within particular types or sub-types of malignancy. The ability to define either consistent changes or sub-types of malignancy is often masked by tumor heterogeneity. To elucidate therapeutic targets in cutaneous squamous cell carcinoma (cSCC), the most frequent skin neoplasm with malignant potential, we have developed an integrated approach to gene expression profiling beginning with primary keratinocytes in culture. Candidate drivers of cSCC development were derived by first defining a set of in vitro cancer genes and then comparing their expression in a range of clinical data sets containing normal skin, cSCC and the benign hyper-proliferative condition psoriasis. A small interfering RNA (siRNA) screen of the resulting 21 upregulated genes has yielded targets capable of reducing xenograft tumor volume in vivo. Small-molecule inhibitors for one target, Polo-like kinase-1 (PLK1), are already in clinical trials for other malignancies, and our data show efficacy in cSCC. Another target, C20orf20, is identified as being overexpressed in cSCC, and siRNA-mediated knockdown induces apoptosis in vitro and reduces tumor growth in vivo. Thus, our approach has shown established and uncharacterized drivers of tumorigenesis with potent efficacy as therapeutic targets for the treatment of cSCC.

Figure 1

cSCC keratinocytes readily form tumors in SCID mice with identical histology to human cSCC. Female SCID Balb/c mice were subcutaneously injected in the right flank with 1–4 × 10⁶ tumor cells mixed with high-concentration Matrigel (Becton Dickinson). Tumor volumes were measured twice a week with calipers and calculated using the formula, V=π4/3((L+W)/4)3, were L is the length and W is the width. (a) Representative growth of a single tumor from eight separate cSCC keratinocyte populations. The average number of days to reach a volume of 100 mm³ from 1–4 separate experiments was as follows: SCCRDEB2 (49 days±5.6 s.d., n=3), SCCIC1 (11.8 days±3.4 s.d., n=12), SCCT2 (59.7 days±8.1 s.d., n=3), SCCT3 (128.3 days±11.7 s.d., n=3) and SCCT8 (65 days±7.1 s.d., n=2). (b) H&E-stained sections of a representative xenograft tumor for each of the six cell populations that showed measurable growth in mice ( × 100 magnification). cSCC, cutaneous squamous cell carcinoma; H&E, hematoxylin and eosin; SCID, severe combined immunodeficient.

Figure 2

Expression profiling of quiescent cultures of early-passage keratinocytes separates cSCC from normal and identifies potential tumor drivers in vivo. (a) Growth rates of keratinocytes used in this study as assessed by a colorimetric assay of mitochondrial dehydrogenase activity when seeded at low density (upper panel) or at confluence (lower panel). Confluent keratinocytes are quiescent after 48 h of culture. RNA for subsequent array experiment was isolated between 48 and 56 h after confluence. (b) An un-supervised clustering dendrogram of in vitro gene expression data generated by BRB-ArrayTools v3.8.1. cSCC keratinocyte samples (red box) cluster independently of non-cSCC keratinocyte samples (blue box). (c) Average cSCC versus normal skin (NS) fold change for all 154 concordantly expressed in vivo cSCC genes plotted against average psoriatic lesional skin versus psoriatic non-lesional skin (NLS) fold change shows a strong overall correlation (r²=0.84) and identifies genes specifically differentially regulated in cSCC. cSCC, cutaneous squamous cell carcinoma; EBKs, primary non-SCC RDEB keratinocytes; NHKs, primary normal human keratinocytes. A full colour version of this figure is available at the Oncogene journal online

Figure 3

PLK1 knockdown and inhibition induces significant G₂/M arrest and apoptosis in cSCC keratinocytes compared with that in normal-proliferating keratinocytes. (a) SCCRDEB2 (SCCK) keratinocytes and primary normal human keratinocytes (NHKs) were transfected with a pool of three separate siRNAs targeting PLK1 and both cell viability and PLK1 protein expression were determined. The percentage of viable cells relative to time 0 (T0) is shown. Whole-cell lysate was extracted from the same SCCRDEB2 keratinocytes or NHKs treated with the same transfection reagent mix at the same time as those used for the viability assessment shown, and PLK1 protein levels were determined by western blotting (lower panel). All results shown represent the mean±s.d.; ***P<0.001 compared with the scrambled control siRNA (SCR) (n=3). (b) The SCCIC1 (SCCK) keratinocytes and NHKs cell viability time course over 72 h of treatment with the PLK1 inhibitors BI 2536 (top panel) and GW843682X (bottom panel). The percentage viable cell number is shown relative to values at T0 (<100% represents a net decline in cell number, >100% represents a net increase in cell number). Representatives of a minimum of three experiments are shown. The results shown represent the mean±s.d., n=3. The NHKs used in each experiment were isolated from different donors. (c) Whole-cell lysate was extracted from SCCIC1 keratinocytes and subjected to western blot analysis of PLK1 protein levels 24 h after exposure to 10 μ GW843682X or 5 μ BI 2536 as indicated. (d) Cell-cycle analysis in SCCIC1 keratinocytes treated either with a pool of three separate PLK1 targeting siRNA (top panel) or with the PLK1 inhibitors GW843682X (10 μ) and BI 2536 (5 μ) (bottom panel), and stained for BrdU and propidium iodide. Results expressed as percentage of cells at the G₀/G₁, S and G₂/M phases of cell cycle 20 or 16 h following treatment, respectively. The results shown are the mean±s.d. of three independent experiments. (e) SCCIC1 keratinocytes transfected with a pool of three separate siRNAs targeting PLK1 or a cell death-positive control siRNA (upper panel), or treated with GW843682X (10 μ) and BI 2536 (5 μ) (lower panel), were assayed for induction of apoptosis using a Cell Death Detection ELISA 24 or 20 h after treatment. The results show the fold increase in absorbance associated with increased cytoplasmic nucleosomes relative to scrambled control non-targeting siRNA or drug vehicle control, respectively. Shown is a representative experiment, with each experiment performed a minimum of three times. The results are the mean±s.d., n=3; *P<0.05, **P<0.01, ***P<0.001 compared with the control. BrdU, 5-Bromodeoxyuridine; cSCC, cutaneous squamous cell carcinoma; PLK, Polo-like kinase; siRNA, small interfering RNA.

Figure 4

C20orf20 knockdown induces apoptosis in cSCC cells without cell-cycle arrest, with no measurable effect on normal keratinocyte growth. (a) SCCIC1 keratinocytes and primary normal human keratinocytes (NHKs) were transfected with a pool of three separate siRNAs targeting C20orf20 (C20) or a scrambled non-targeting control siRNA (Scr), and cell viability was assessed by MTS assay (left panel). The results shown are 48 h after seeding of transfected cells and expressed relative to T0. The relative expression of C20orf20 following siRNA transfection was assessed by reverse transcription–real-time quantitative PCR on the same cells treated with the same transfection reagent mix at the same time as those used for the viability assessment shown, with all data shown relative to the NHK scrambled non-targeting control (right panel). (b) SCCIC1 keratinocytes and HCT116 colorectal cancer cells were transfected with a scrambled non-targeting control, a cell death-positive control or a pool of three separate C20orf20 targeting siRNA, and apoptosis was assessed 24 h later by a Cell Death Detection ELISA. The results show the fold increase in absorbance associated with increased cytoplasmic nucleosomes relative to non-targeting siRNA. Shown is a representative experiment, with each experiment performed a minimum of three times. The results are the mean±s.d., n=3; **P<0.01, ***P<0.001 compared with the control. (c) Cell-cycle analysis in SCCIC1 keratinocytes treated with C20orf20 siRNA. Cells were incubated for 20 h after transfection and labeled with BrdU and propidium iodide. Results expressed as percentage of cells at the G₀/G₁, S and G₂/M phases. The results shown are the mean ±s.d. of three independent experiments. BrdU, 5-Bromodeoxyuridine; cSCC, cutaneous squamous cell carcinoma; siRNA, small interfering RNA.

Figure 5

PLK1 inhibition and C20orf20 knockdown reduces tumor growth in vivo. (a) Top panel: SCCIC1 tumors treated with the PLK1 inhibitor BI 2536. Treatment group tumors (n=3) were injected with BI 2536 at a dose of 25 mg/kg, six times over 2 weeks (arrows). The control group (n=2) was injected with the vehicle following the same schedule. Bottom panel: SCCIC1 tumors treated with C20orf20 siRNA. Treatment group (n=3) tumors were injected with 580 pmol of a pool of three separate C20orf20 targeting siRNA duplexes in 100 μl of phosphate-buffered saline. The control group (n=3) was injected with 580 pmol of a scrambled non-targeting control siRNA duplex in 100 μl of phosphate-buffered saline. The animals were killed 2 days after the last treatment. (b) A representative H&E-stained section (top) and a keratin-immunostained section (bottom) of control (left) and BI 2536-treated (right) SCCIC1 tumors after 2 weeks of treatment. Although no difference in tumor volume between the control and the treated group was seen at this time point, histology shows that the BI 2536-treated tumors consisted of mainly inert material (keratin) without discernable SCCIC1 tumor cells, whereas control tumors contained numerous SCCIC1 cells ( × 100 magnification). (c) Photographs from the experiment shown in panel a of vehicle- (top left, tumor bisected showing both halves delineated by the dashed line) and BI 2536-treated (top right) SCCIC1 tumors. The largest SCCIC1 tumor treated with C20orf20 siRNA in the experiment shown in panel a (bottom right, tumor bisected, showing both halves delineated by a dashed line) is hollow in appearance compared with the scrambled non-targeting control siRNA-treated tumor (bottom left). H&E, hematoxylin and eosin; PLK, Polo-like kinase; siRNA, small interfering RNA.