RSK2 expression correlates with cell invasive ability of diverse human HNSCC cell lines and HNSCC progression.
We performed an in vitro Matrigel invasion assay, using diverse human HNSCC cell lines. Based on the differential invasive ability, these cell lines were divided into 2 groups: a poorly invasive group that includes Tu686, 37A, Tu212, and 686LN and a highly invasive group that includes M4e, 37B, 212LN, 4A, 4B, and 886LN (Figure A). We found that RSK2 was overexpressed and activated, as assessed by phosphorylation at Ser386, in the highly invasive cell lines, including M4e, 37B, 212LN, 4A, 4B, and 886LN, compared with the poorly invasive cell lines, including Tu686, 37A, Tu212, and 686LN (Figure B). These data suggest that RSK2 expression may promote HNSCC cell invasion and tumor metastasis.
RSK2 is overexpressed in a group of highly invasive human HNSCC metastatic cell lines, and the pattern of RSK2 expression correlates with HNSCC cancer progression.
We next conducted a study to detect RSK2 expression by an immunohistochemistry (IHC) assay, using primary human HNSCC patient tissue samples. We first characterized the RSK2 antibody (Novus Biologicals) using RSK2-negative Tu212 cells and RSK2-positive 212LN cells that were embedded in paraffin (Supplemental Figure 1A; supplemental material available online with this article; doi:
). Positive Western blot and IHC staining of RSK2 was observed in 212LN cells but not in Tu212 cells. Moreover, we evaluated this antibody using primary tumor tissue samples from xenografted mice injected with either control M4e-pLKO.1 cells or M4e-pLKO.1-RSK2 shRNA cells with stable knockdown of RSK2 (described below). Positive Western blot and IHC staining of RSK2 was observed in tumor tissue samples derived from control M4e-pLKO.1 cells but not in those from M4e-pLKO.1-RSK2 shRNA cells (Supplemental Figure 1B).
Tumor and LN specimens representing 3 categories were evaluated, including primary tumors from patients with nonmetastatic disease (Tu–Met), primary tumors from patients with metastatic HNSCC (Tu+Met), and paired metastatic LN (LN+Met) samples from the same patients. As shown in Figure C, positive staining of RSK2 was determined by the IHC signal intensity (scored as 0–3+) in the cytoplasm in a percentage of tumor cells. Table presents the summarized data, with statistical analysis showing that the percentages of RSK2-positive cases (scored 1+, 2+, or 3+) are significantly higher in the paired tissue samples of primary tumor (Tu+Met) and metastatic LNs (LN+Met) from patients with metastatic HNSCC than the primary tumor specimens (Tu–Met) from patients with nonmetastatic disease, with P values of 0.007 and less than 0.0001, respectively. Together, these results support our hypothesis that the RSK2 expression pattern correlates with human head and neck cancer metastatic progression. Positive staining of RSK2 in some cases in the group of nonmetastatic primary tumors (Tu–Met) also suggests that RSK2 expression may already be positively selected for, even at the primary tumor stage prior to onset of metastasis.
IHC analysis of primary tissue samples from patients with HNSCC
RSK2 promotes cell invasion in HNSCC cells.
To further substantiate the role of RSK2 in HNSCC metastasis, we next determined whether RSK2 overexpression could confer invasive potential to the poorly invasive human HNSCC cell lines, including 686LN, Tu212, and 37A, that lack high levels of RSK2 expression. In an in vitro Matrigel invasion assay, we observed that transiently enforced RSK2 expression in these 3 independent cell lines (Figure A) significantly enhanced the invasive ability of these cells (Figure A). We then assessed the impact of targeting RSK2 on invasion of HNSCC cells. We first examined the effects of inhibiting RSK2 kinase activity by using highly specific RSK inhibitors. RSK inhibitor-fmk [RSKI-fmk; 1-(4-amino-7-(3-hydroxypropyl)-5-p
) is a fluoromethylketone molecule that was designed to specifically exploit 2 selectivity filters of RSK. RSKI-fmk potently inactivates the C-terminal auto-kinase domain activity of RSK1 and RSK2 with high specificity in mammalian cells (14
). As shown in Figure B, treatment with RSKI-fmk effectively inhibited RSK2 kinase activity in 3 RSK2-expressing HNSCC cell lines, including M4e, 212LN, and 37B, as assessed by phosphorylation at Ser386, an index of RSK2 activation. Moreover, upon RSKI-fmk treatment, these cells demonstrated a significant attenuation of invasion (Figure , C and D). Similar results were obtained by using another RSK inhibitor, BI-D1870, which was derived from dihydropteridinones and identified as a highly specific and potent inhibitor of RSK N-terminal trans-kinase domain in kinase selectivity screening (Figure E) (16
). Thus, targeting RSK2 by 2 distinct inhibitors, which differentially inhibit RSK2 kinase activity, results in marked reduction of HNSCC cell invasive ability. However, such decreased cell invasive ability, associated with inhibition of RSK2, was not a consequence of reduced proliferation, because treatment with either RSKI-fmk or BI-D1870 did not significantly affect the proliferation rate of M4e, 212LN, and 37B cells (Figure F).
Expression of RSK2 promotes in vitro HNSCC cell invasion.
We also examined the effect of RNAi-mediated RSK2 knockdown on invasion of M4e, 212LN, and 37B cells. We first used pools of siRNA specifically targeting RSK1 or RSK2 and a non-specific siRNA as a negative control. Both RSK1 and RSK2 siRNAs were highly specific in decreasing their respective target protein expression in various HNSCC cell lines (Figure A). Transient transfection of RSK2-specific siRNA induced significant inhibition of invasion of M4e, 212LN, and 37B cells into matrix proteins in the Matrigel assay (Figure B), compared with cells transfected with nonspecific siRNA. In contrast, RSK1 siRNA failed to induce inhibition of cell invasion in these HNSCC cells (Figure B). Transient transfection with nonspecific RSK1 or RSK2 siRNAs did not significantly affect the proliferation rate of M4e, 212LN, and 37B cells (Figure C; in parallel, Western blot control is shown in Supplemental Figure 2A). These findings indicate that RSK2 but not RSK1 is involved in regulation of HNSCC cell invasion. Similar results were obtained using a lentiviral shRNA vector, pLKO.1-RSK2 shRNA, which was documented to stably reduce RSK2 protein expression by approximately 90% in distinct HNSCC cell lines (Figure D). Stably targeted downregulation of RSK2 using this lentiviral vector in M4e, 212LN, and 37B cells resulted in significant reduction of the invasive ability of these cells in vitro (Figure E) but did not significantly affect the proliferation rate of M4e, 212LN, and 37B cells (Figure F; in parallel, Western blot control is shown in Supplemental Figure 2B). An additional control experiment showed that stable transduction of M4e and 212LN cells with lentiviral vectors harboring scrambled shRNA or shRNA targeting GFP did not significantly affect the proliferation rate of these cells, nor did it alter the protein expression levels of RSK2 or β-actin in M4e or 212LN cells (Supplemental Figure 3 and 4).
Targeted downregulation of RSK2 attenuates HNSCC cell invasive ability.
Targeted downregulation of RSK2 by shRNA in metastatic M4e cells inhibits development of LNM in a xenograft mouse model.
We next assessed the effect of knockdown of RSK2 by shRNA on the HNSCC cell metastatic potential in vivo, using a LNM xenograft mouse model (17
). We used M4e cells that were demonstrated to be highly metastatic in this model (17
). M4e cells, which were stably transduced with the lentiviral shRNA vector targeting RSK2 (pLKO.1-RSK2 shRNA) or an empty control vector (pLKO.1) (Figure A), were injected into nude mice. The mice were sacrificed on day 21, which was an endpoint determined based on our experience that M4e cells induced tumor development as well as detectable LNM in 3 weeks (data not shown). Compared with control M4e cells transduced with empty vector, M4e-pLKO.1-RSK2 shRNA cells, with stable knockdown of RSK2, showed a marked attenuation of LNM, which was characterized by the number of LNs invaded by M4e cells (Table ), determined by IHC staining of human vimentin, a mesenchymal cell marker that is expressed in the human metastatic M4e cells but not in mouse LNs (Figure B).
RNAi-mediated stable knockdown of RSK2 significantly attenuates the metastatic potential of M4e cells in a xenograft nude mouse model.
M4e-pLKO.1-RSK2 shRNA cells, with stable knockdown of RSK2, showed a marked attenuation of LNM
In summary, these loss-of-function and gain-of-function studies in cells and mice support the view that continued RSK2 expression is important for maintaining the invasive and metastatic potential of HNSCC cells.
RNAi-mediated RSK2 knockdown does not attenuate proliferation and tumor formation of metastatic HNSCC cells.
We next examined the possibility of whether the RSK2-dependent metastatic phenotype in vivo might be a consequence of increased proliferation. In the LNM xenograft nude mouse assay, no significant difference in the size of primary tumors was detected between the 2 groups of mice injected with either M4e-pLKO.1 or M4e-pLKO.1-RSK2 shRNA cells (Figure C). Moreover, stable knockdown of RSK2 in M4e-pLKO.1-RSK2 shRNA cells did not affect cell proliferation, which was assessed using the percentage of cells with positive staining of Ki-67 in each primary tumor tissue sample, when compared with control M4e-pLKO.1 cells (Table and Figure D). Together, these in vivo data demonstrate that RSK2 possesses intrinsic prometastatic activity in HNSCC cells, which is not a consequence of enhanced proliferation.
Stable knockdown of RSK2 in M4e cells did not attenuate cell proliferation in derived tumors
RSK2 promotes metastasis in HNSCC cells by regulating multiple prometastatic protein factors.
To explore the molecular mechanism underlying RSK2-enhanced metastasis, we surveyed potential links between RSK2 and some signaling molecules of known relevance to cell invasion and tumor metastasis. However, RNAi-mediated RSK2 knockdown in metastatic M4e cells did not affect the phosphorylation levels of ERK, AKT, and STAT3 (Figure A). To comprehensively find mechanistic insight into the role of RSK2 in HNSCC metastasis, we performed a phospho-proteomics–based study using a phospho-antibody microarray (Full Moon BioSystems Inc.), which provides a high-throughput platform for efficient protein phosphorylation status profiling, with detection and analysis of phosphorylation events at specific sites to identify RSK2 downstream substrates/effectors that regulate metastasis. The experiment was performed using the MAPK Pathway Phosphorylation Antibody Array, since RSK2 is a substrate of ERK. Cell lysates obtained from M4e-pLKO.1, and M4e-pLKO.1-RSK2 shRNA cells were applied. We identified a spectrum of proteins whose phosphorylation levels were decreased more than 15%, with low values of 95% CI in M4e cells when RSK2 was stably knocked down. Many of these proteins, when phosphorylated, are important for cell migration, invasion, and tumor metastasis. These prometastatic proteins include c-Jun, CREB, Elk-1, focal adhesion kinase (FAK), Hsp27, IRS-1, JunB, c-MET, and Stathmin (Table and Supplemental Table 1).
Targeting RSK2 by shRNA leads to decreased phosphorylation levels of multiple prometastatic protein factors, including tyrosine kinases, c-MET and FAK, and RSK2 phosphorylation substrates, CREB and Hsp27.
Protein factors whose phosphorylation states decreased in M4e cells when RSK2 was stably knocked down by shRNA
Among these proteins, we confirmed by immunoblotting that targeted downregulation of RSK2 by shRNA resulted in reduced phosphorylation and activation levels of the receptor tyrosine kinase c-MET in M4e cells and FAK in 212LN and 37B cells (Figure B). Although RSK2, as a serine/threonine kinase, is unlikely to directly regulate activation of these 2 prometastatic tyrosine kinases by phosphorylation, a decrease in FAK and c-MET activation upon RSK2 downregulation demonstrates the reprogramming of the HNSCC prometastatic signaling network upon RSK2 knockdown.
RSK2 phosphorylates and activates prometastatic CREB and Hsp27.
To characterize the signaling properties of RSK2 in HNSCC cell invasion, we next focused on 2 potential substrates/effectors of RSK2, including a known RSK2 substrate, CREB, and Hsp27. RSK2 has been demonstrated to regulate and activate CREB by phosphorylating Ser133 (18
). CREB is a transcription factor whose signaling is implicated in tumor growth and metastasis in human prostate cancer (PCa) and melanoma (19
Hsp27 regulates actin dynamics (23
) and has been found to be overexpressed in many human cancers. Hsp27 is regulated by phosphorylation at Ser15, Ser78, and Ser82. Phosphorylation of Hsp27 is associated with tumor cell migration and invasion and correlates with LN positivity in breast cancer (24
). We first determined whether RSK2 directly phosphorylates Hsp27 in an in vitro kinase assay, in which purified recombinant Hsp27 (rHsp27) WT and individual S15A, S78A, or S82A mutant proteins were incubated with active recombinant RSK2 (rRSK2). As shown in Figure C, the immunoblotting results using the specific phospho-Hsp27 antibodies (pS78 and pS82) demonstrate that rHsp27 WT, but not the S78A or S82A mutants, was phosphorylated at Ser78 or Ser82 by RSK2, respectively. In contrast, immunoblotting using a specific phospho-Hsp27 antibody (pS15; CST) showed that both rHsp27 WT and S15A mutant proteins were not phosphorylated at Ser15 by rRSK2 (Figure C). Further, in vitro kinase assays using another Hsp27 phospho-Ser15 antibody (Santa Cruz Biotechnology) and mass spectrometry–based studies confirmed that Ser15 of Hsp27 is not phosphorylated by RSK2 (data not shown). These data suggest that to our knowledge Hsp27 is a newly identified phosphorylation substrate of RSK2.
As shown in Figure D, CREB S133 phosphorylation was markedly reduced in metastatic M4e, 212LN, and 37B cells upon RNAi-mediated RSK2 knockdown. Similarly, stable RSK2 knockdown in M4e and 212LN cells resulted in reduced Hsp27 S78 and S82 phosphorylation. However, RSK2 knockdown did not affect phosphorylation levels of Hsp27 S78 and S82 in 37B cells (Figure , D and E, respectively). Such differences may be due to disparities in the cellular contexts of distinct cell lines (discussed below).
Expression and phosphorylation levels of CREB and Hsp27 are required for RSK2-mediated pro-invasive ability of HNSCC cells.
We also examined the effect of RNAi-mediated knockdown of CREB and Hsp27 on invasion of the RSK2-expressing, metastatic M4e cells. Both CREB and Hsp27 shRNAs were specific in decreasing their respective target protein expression in M4e cells (Figure A). Stably targeted downregulation of CREB or Hsp27, using lentiviral vectors containing distinct shRNAs in M4e cells, resulted in significant reduction of invasive ability in vitro (Figure B) but did not significantly affect the proliferation rate of these cells (Figure C).
RSK2 promotes HNSCC cell invasion through phosphorylation and activation of the downstream substrate CREB.
We also found that enforced expression of RSK2 enhanced the invasive ability of Tu212 cells, whereas shRNA-mediated knockdown of CREB (Figure D) significantly attenuated such RSK2 expression–induced cell invasion. Moreover, stable expression of CREB WT resulted in significantly increased cell invasive ability of 686LN (Figure E) and Tu212 (Figure F) cells, and cells expressing a phospho-mimetic mutant CREB S133D demonstrated further increased cell invasive ability. In contrast, overexpressing the phospho-deficient mutant CREB S133A similarly resulted in increased cell invasion of 686LN and Tu212 cells, as did CREB WT (Figure , E and F, respectively), compared with control cells, but failed to further enhance the cell invasive ability like the phospho-mimetic mutants. These results suggest that expression of CREB has both S133 phosphorylation-dependent and -independent effects on HNSCC cell invasion. In poorly invasive 686LN and Tu212 cells that lack high levels of RSK2 expression, only the phospho-mimetic CREB S133D, but not the phospho-deficient CREB S133A mutant, further increased cell invasive ability, compared with CREB WT. This suggests that the S133 phosphorylation-dependent pro-invasive effects of CREB depend on RSK2 expression and activation.
RSK2 promotes cell invasion by phosphorylating Hsp27 to regulate stabilization of actin filaments in HNSCC cells.
Similarly, shRNA-mediated knockdown of Hsp27 significantly attenuated Tu212 cell invasion conferred by enforced expression of RSK2 (Figure A). Stable expression of Hsp27 WT led to increased cell invasion of poorly invasive Tu212 cells (Figure B) but not 686LN cells (Figure B). Interestingly, expression of Hsp27 phospho-mimetic mutants S78D or S82D did not lead to enhanced cell invasive capability compared with cells expressing Hsp27 WT, whereas expression of the double-mutant Hsp27 S78D/S82D significantly potentiated cell invasion of Tu212 (Figure B) and 686LN (Figure B). This result demonstrates that phosphorylation of Hsp27 at both Ser78 and Ser82 by RSK2 is crucial for its proinvasive function in HNSCC cells, while phosphorylation of Ser78 or Ser82 alone is insufficient to activate Hsp27. In contrast, overexpression of the phospho-deficient mutants Hsp27 S78A/S82A similarly resulted in increased cell invasion of 686LN and Tu212 cells to a similar degree as Hsp27 WT cells, compared with control cells, but failed to further enhance the cell invasive ability, like the phospho-mimetic mutant Hsp27 S78D/S82D (Figure B). Thus, these results together also implicate that Hsp27 has both S78/S82 phosphorylation-dependent and -independent effects on HNSCC cell invasion. In poorly invasive Tu212 cells that lack high expression levels of RSK2, only the phospho-mimetic double-mutant Hsp27 S78D/S82D, but not the single mutants S78D and S82D or the phospho-deficient S78A/S82A mutant, further increased cell invasive ability compared with Hsp27 WT (Figure B). This suggests that the S78/S82 phosphorylation-dependent proinvasive effects of Hsp27 depend on RSK2 expression and activation. However, in 686LN cells, Hsp27 may only have the S78/S82 phosphorylation-dependent effect because overexpression of Hsp27 WT in these cells did not result in increased cell invasion compared with control cells (Figure B).
RSK2 promotes stabilization of actin filaments in HNSCC cells through phosphorylation and activation of Hsp27.
We also found that stable expression of the Hsp27 phospho-mimetic mutant S78D/S82D, but not WT or the phospho-deficient S78A/S82A mutant, partially rescued cell invasion in M4e cells with stable knockdown of RSK2 (Figure C). However, neither RNAi-mediated stable knockdown of RSK2 nor stable expression of Hsp27 WT, S78D/S82D, or S78A/S82A variants significantly affected the proliferation rate of M4e cells (Supplemental Figure 5).
Hsp27 has been implicated in regulation of cytoskeleton dynamics by stabilizing actin filaments (26
). To determine whether RSK2 regulates actin filaments, we examined the integrity of actin filaments in 212LN and M4e cells with stable knockdown of RSK2. We found that actin filaments were well organized and distributed evenly throughout the control 212LN and M4e cells transduced with empty vector. In contrast, knockdown of RSK2 induced the disruption of actin filaments and redistribution of actin to the cell membrane (Figure D). However, stable expression of the Hsp27 phospho-mimetic mutant S78D/S82D, but not WT or the phospho-deficient S78A/S82A mutant, rescued the formation of actin filaments in M4e cells with stable knockdown of RSK2 (Figure D), which correlates with rescued M4e cell invasion, as shown in Figure C. These data demonstrate that RSK2 regulates stabilization of actin filaments in HNSCC cells through phosphorylation of Hsp27.