RSK2 as a key regulator in human skin cancer

Abstract

Our previous report demonstrated that RSK2 plays an important role in cell proliferation and transformation induced by tumor promoters such as epidermal growth factor mediated through the N-terminal kinase domain of RSK2 in JB6 Cl41 mouse skin epidermal cells in vitro. However, no direct evidence has been reported regarding the relationship of RSK2 activity and human skin cancer. To elucidate the relationship of RSK2 activity and human skin cancer, we examined the effect of knocking down RSK2 expression on epidermal growth factor-induced anchorage-independent transformation in the premalignant HaCaT human skin keratinocyte cell line and on soft agar colony growth of SK-MEL-28 malignant melanoma cells. We found that the phosphorylated protein levels of RSK2 were enhanced in cancer tissues compared with normal tissues in a human skin cancer tissue array. We found that UVB stimulation induced increased in not only the total and phosphorylated protein levels of ERKs and RSK2 but also the nuclear localization and gene expression of RSK2. RSK2 knockdown inhibited proliferation and anchorage-independent transformation of HaCaT cells and soft agar colony growth of malignant melanoma cells. Moreover, RSK2(-/-) mouse embryonic fibroblast (MEF) showed enhanced sub-G(1) accumulation induced by UVB stimulation compared with RSK2(+/+) MEFs, indicating that RSK2 might play an important role in tolerance against stress associated with ultraviolet. Importantly, activated RSK2 protein levels were highly abundant in human skin cancer tissues compared with matched skin normal tissues. Taken together, our results demonstrated that RSK2 plays a key role in neoplastic transformation of human skin cells and in skin cancer growth.

Fig. 1.

UVB activates ERKs-RSK2 signaling. (A) Immunofluorescence of total RSK2 proteins in normal human skin tissues. Normal human skin tissues were used to visualize total RSK2 proteins using an RSK2-specific primary antibody and an Alexa568-conjugated secondary antibody. Optical sections showing total RSK2 protein levels were captured by laser scanning confocal microscopy as described in Materials and methods. (B) Activation profile of ERKs-RSK2 signaling induced by UVB exposure. HaCaT human skin keratinocytes (4×10⁶) were seeded in 100mm culture dishes, incubated for 24h, starved overnight, and then stimulated with UVB (4 kJ/m²). The cells were harvested and proteins extracted. Activation of ERKs-RSK2 signaling was analyzed by western blotting using specific antibodies as indicated. β-Actin was used as an internal control to monitor equal protein loading. (C) UVB stimulates increased rsk2 mRNA expression. HaCaT human skin keratinocytes (4×10⁶) were seeded, cultured for 24h, starved overnight and stimulated with UVB (4 kJ/m²). Total RNA was extracted at the indicated time point and rsk2 mRNA levels were analyzed by real-time RT–PCR as described in Materials and methods. Data are represented as means ± SD of values obtained from two independent triplicate experiments and significant differences were calculated using the Student’s t-test (*P < 0.05, **P < 0.001). (D) UVB induces nuclear localization of RSK2. HaCaT human skin keratinocytes (2×10⁴) were seeded into 2-chamber slides, cultured for 24h, and then serum-starved overnight. Cells were stimulated with UVB (4 kJ/m²), fixed with 4% formalin for 15min and permeabilized with 0.5% triton X-100 in 1× PBS. Nuclear localization of RSK2 was observed by fluorescence microscope (×200) using a phospho-RSK2 (T577) primary antibody and an Alexa 488-conjugated secondary antibody. The fluorescence intensity was measured by the Image J computer program (v.1.45) and normalized by fluorescence of DAPI intensity. Data are represented as means ± SD of values obtained from 20 randomly selected cells and significant differences were calculated using the Student’s t-test (*P < 0.05, **P < 0.005).

Fig. 2.

RSK2 enhances tolerance against UVB-induced stress. (A) RSK2 wild-type (RSK2^+/+) and deficient (RSK2^–/–) MEFs were treated with UVB (4 kJ/m²) and the sub-G₁ population was analyzed by flow cytometry at the indicated time point. Data are represented as means ± SD of values obtained from an experiment with triplicate samples and significant differences were calculated using the Student’s t-test (*P < 0.05, **P < 0.005). (B) HaCaT human skin keratinocytes (4×10⁶) were seeded in a 100mm dish, cultured for 24h, starved, stimulated with UVB (4 kJ/m²), and then harvested at the indicated time point. The cytosolic and nuclear proteins were extracted using NE/PER nuclear and cytoplasmic extraction reagents. Activated and total RSK2 protein levels were visualized using specific antibodies as indicated. Lamin B and α-tubulin were used as nuclear and cytoplasmic marker proteins, respectively. (C) UVB stimulation induces increased RSK2 protein levels in various skin cell lines. HaCaT, N/TERT-1, SCC-13 and SK-MEL-28 cells were seeded, starved, stimulated with UVB (4 kJ/m²) and cultured for 60min in a 37^°C/5% CO₂ incubator. The proteins were extracted and then activated and total RSK2 protein levels were visualized by western blotting using specific antibodies as indicated. β-Actin was used as an internal control to monitor equal protein loading. (D) HaCaT, N/TERT-1, SCC-13 and SK-MEL-28 cells (2×10⁴) were seeded in 4-chamber slides, starved for 24h, stimulated with UVB and cultured for 60min in a 37^°C/5% CO₂ incubator. The cells were fixed, permeabilized, triple-stained with Alexa-488 to detect total RSK2, Alexa-568 to detect phosphor-RSK2 (T577) and DAPI for nuclear staining. Activated and total RSK2 protein levels were visualized under a laser scanning confocal microscope using specific antibodies as indicated.

Fig. 3.

RSK2 knockdown inhibits normal human skin and cancer cell proliferation. (A) Endogenous RSK2 protein levels in normal human skin HaCaT, N/TERT-1 keratinocytes, human skin SCC-13 and SK-MEL-28 cancer cells. Cells were cultured with complete cell culture media and proteins were extracted. The RSK2 protein levels were analyzed by western blotting using RSK2-specific primary antibody and a horseradish peroxidase-conjugated secondary antibody. β-Actin was used as an internal control to monitor equal protein loading. (B) Endogenous RSK2 expression is silenced by infection with sh-RSK2 in HaCaT, N/TERT-1, SCC-13 and SK-MEL-28 cells. Knockdown efficiency was analyzed by western blotting using an RSK2-specific primary antibody and a horseradish peroxidase-conjugated secondary antibody. β-Actin was used as an internal control to monitor equal protein loading. (C) The cells from (B) were used for a proliferation assay. Cells (1×10³) were seeded into 96-well plates and proliferation was measured by MTS assay at 24h intervals up to 96h. Data are represented as means ± SD of values obtained from six experiments and significant differences were calculated using the Student’s t-test (*P < 0.05, ** P < 0.005).

Fig. 4.

Knockdown of RSK2 suppresses EGF-induced anchorage-independent cell transformation and colony growth in soft agar. (A) Suppression of anchorage-independent transformation of HaCaT cells by knocking down endogenous RSK2. Cells stably expressing sh-mock or sh-RSK2 were cultured and subjected to an EGF-induced anchorage-independent cell transformation assay. Cells (8×10³) were exposed to EGF (10ng/ml) in 1ml of 0.3% Basal Medium Eagle agar containing 10% FBS. The cultures were maintained in a 37°C, 5% CO₂ incubator for 14 days and then colonies were counted using a microscope and the Image-Pro PLUS (v.6.2) computer software program. Data are represented as means ± SD of values from triplicate experiments and statistical significance was determined using the Student’s t-test (*P < 0.001). (B) Colony growth in soft agar is inhibited by knocking down endogenous RSK2. SK-MEL-28 cells stably expressing sh-mock or sh-RSK2 were tested for colony growth under anchorage-independent conditions. Cells (8×10³) were mixed with 0.3% top agar in complete cell culture medium. The cultures were maintained in a 37°C, 5% CO₂ incubator for 10 days and then colonies were counted using a microscope and the Image-Pro PLUS (v.6.2) computer software program. Data are represented as means ± SD of triplicate values and statistical significance was determined using the Student’s t-test (*P < 0.001).

Fig. 5.

Tissue array analysis of RSK2 in solid human skin cancer and normal skin tissues. (A) Activated RSK2 protein levels are higher in cancer tissues compared with normal tissues. An analysis of activated RSK2 was performed using a human skin tissue array that included matched normal and cancer tissues. Phosphorylated RSK2 was detected using an antibody against phospho-RSK2 (T577) and an Alexa 488-conjugated secondary antibody as described in Materials and methods. The light intensity of each sample was estimated using the Image J computer program (v.1.45). The overall average density of all samples is shown (left panel) along with the density of each individual sample (right panel). The fluorescence intensity of the individual sample was obtained using the Image J computer software program (v.1.45). (B–C) The 70 core samples of skin cancer tissues were classified into subclasses of skin cancer categories, including SCC, BCC and MM. The fluorescence light intensity of the activated RSK2 protein and total RSK2 protein levels were re-evaluated. The average density of each subclass is shown in Supplementary Figure 1A (total RSK2) and 1B [phospho-RSK2 [T577]), available at Carcinogenesis Online along with the density of each individual sample for total RSK2 (B) and activated RSK2 (C). Significant differences in A–C were evaluated using the Student’s t-test as described previously (7).

Fig. 6.

The activated RSK2 protein level is higher in cancer tissues compared with matched normal tissues. (A) The levels of activated RSK2 proteins were compared with matched human normal and cancer tissues, including SCC, chronic inflammation of fibrous tissue and MM. Representative samples are shown. (B) The relative light intensity of individual activated RSK2 in cancer tissues compared with matched each normal tissue sample is shown. (C) Elevation of the activated RSK2 protein levels in skin cancer tissue. A matched human skin cancer tissue and normal tissue in (A) were magnified to confirm whether activated RSK2 protein levels were accumulated in nucleus skin cancer tissue. The nucleus was stained with DAPI, phosphorylated RSK2 (T577) was stained with Alexa-488 and nuclear localization of RSK2 was confirmed by merging of DAPI and Alexa-488 as indicated.