RSK Promotes Prostate Cancer Progression in Bone Through ING3

Published OnlineFirst September 4, 2014; DOI: 10.1158/1541-7786.MCR-14-0384-T

Oncogenes and Tumor Suppressors Molecular Cancer Research RSK Promotes Prostate Cancer Progression in Bone through ING3, CKAP2, and PTK6-Mediated Cell Survival Guoyu Yu1, Yu-Chen Lee1, Chien-Jui Cheng2,3, Chuan-Fen Wu4, Jian H. Song5, Gary E. Gallick5, Li-Yuan Yu-Lee6, Jian Kuang4, and Sue-Hwa Lin1,5

Abstract

Prostate cancer has a proclivity to metastasize to bone. The determined that injecting C4-2B4/RSK cells into mouse femurs mechanism by which prostate cancer cells are able to survive and enhanced their progression in bone compared with control cells. progress in the bone microenvironment is not clear. Identiﬁcation In PC3-mm2 cells, knockdown of RSK1 (RPS6KA1), the predom- of molecules that play critical roles in the progression of prostate inant RSK isoform, but not RSK2 (RPS6KA2) alone, decreased cancer in bone will provide essential targets for therapy. Ribo- anchorage-independent growth in vitro and reduced tumor pro- somal S6 protein kinases (RSK) have been shown to mediate gression in bone and tumor-induced bone remodeling in vivo. many cellular functions critical for cancer progression. Whether Mechanistic studies showed that RSK regulates anchorage-inde- RSK plays a role in the progression of prostate cancer in bone is pendent growth through transcriptional regulation of factors that unknown. IHC analysis of human prostate cancer specimens modulate cell survival, including ING3, CKAP2, and PTK6. showed increased phosphorylation of RSK in the nucleus of Together, these data provide strong evidence that RSK is an prostate cancer cells in a signiﬁcant fraction of human prostate important driver in prostate cancer progression in bone. cancer bone metastasis specimens, compared with the primary site or lymph node metastasis. Expression of constitutively active Implications: RSK, an important driver in prostate cancer pro- myristylated RSK in C4-2B4 cells (C4-2B4/RSK) increased their gression in bone, has promising potential as a therapeutic target survival and anchorage-independent growth compared with C4- for prostate cancer bone metastasis. Mol Cancer Res; 13(2); 348–57. 2B4/vector cells. Using an orthotopic bone injection model, it was 2014 AACR.

Introduction play critical roles in the progression of prostate cancer in bone will provide targets for therapy. Prostate cancer is the second leading cause of cancer-related Ribosomal S6 protein kinase (RSK) is a family of signal death in men in the United States. Mortality from prostate cancer transducing Ser/Thr kinases. Four isoforms, RSK1–4, have been is due mainly to development of metastasis in bone. Prostate reported in mammalian cells (for review, see refs. 1–4). The cancer has a proclivity to metastasize to bone. One critical feature best functionally characterized isoforms are RSK1 and RSK2. for metastatic prostate cancer cells to colonize in bone is to survive Each RSK isoform contains two nonidentical kinase domains, in the bone microenvironment. The mechanism by which pros- one at the N-terminus and one at the C-terminus. Phosphor- tate cancer cells are able to survive and progress within the bone ylation of RSKs at Ser/Thr, which occurs at multiple sites, is microenvironment is not clear. Identiﬁcation of molecules that required for RSK activation (4) and the N-terminal kinase domain is primarily responsible for substrate phosphorylation (5). RSKs phosphorylate many proteins, both cytosolic and 1Department of Translational Molecular Pathology, The University of nuclear (2). The many effects of RSKs on various proteins may Texas M. D. Anderson Cancer Center, Houston, Texas. 2Department of contribute to the observations that RSKs mediate wide-ranging Pathology, College of Medicine, Taipei Medical University, Taipei, cellular processes, including proliferation (6–8), migration (9), Taiwan. 3Department of Pathology,Taipei Medical University Hospital, TaipeiMedical University,Taipei,Taiwan. 4Department of Experimental and invasion (1). Therapeutics, The University of Texas M. D. Anderson Cancer Center, Expression of RSK1 and 2 proteins, analyzed by Western blot 5 Houston, Texas. Department of Genitourinary Medical Oncology,The analysis, has been previously shown to increase in prostate cancer University of Texas M. D. Anderson Cancer Center, Houston, Texas. 6Department of Medicine, Baylor College of Medicine, Houston,Texas. when the cancer is localized in the primary site (8). However, whether expression of RSKs is increased in bone metastases is Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). unknown, likely due to the lack of suitable RSK antibody for IHC analysis. Clark and colleagues (8) also showed that RSK inhibition G. Yu, Y.-C. Lee, and C.-J. Cheng contributed equally to this article. decreases the proliferation of cancer cells, including LNCaP and Corresponding Author: Sue-Hwa Lin, The University of Texas M.D. Anderson PC3 prostate cancer cells and MCF-7 breast cancer cells, but not Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794- normal breast epithelial cells MCF-10A (8). These observations 1559; Fax: 713-794-4672; E-mail: [email protected] suggest that RSKs are involved in prostate cancer progression. doi: 10.1158/1541-7786.MCR-14-0384-T Whether RSKs play a role in prostate cancer bone metastasis is 2014 American Association for Cancer Research. unknown.

348 Mol Cancer Res; 13(2) February 2015

RSK in Prostate Cancer Bone Metastasis

In this study, we examined the role of RSKs in prostate cancer Knockdown of RSK1 or RSK2 in PC3-mm2 cells bone metastasis. Our studies showed that expression of RSKs in Lentivirus-expressing shRNAs were generated by cotransfecting prostate cancer cells increases cell survival and anchorage-inde- RSK1 shRNA or RSK2 shRNA plasmids with pCMV-dR8.2 dvpr pendent growth in vitro and enhances prostate cancer progression and pCMV-VSVG packaging plasmids into 293FT cells. PC3- in bone in vivo. mm2-LT cells transduced with pLKO or pLKO-shRSK1 vectors were selected by 5 mg/mL puromycin to generate PC3-pLKO and Materials and Methods PC3-shRSK1, respectively. RSK2 knockdown PC3-mm2 cells were generated similarly using pGIPZ-shRSK2. To generate PC3- Materials shRSK1/2 double knockdown cells, PC3-shRSK1 cells were C4-2B4-LT and PC3-mm2-LT, expressing luciferase and red infected with lentivirus from pGIPZ-shRSK2 and the cells selected fl uorescence protein Tomato, were generated as described previ- by FACS through GFP expressed from pGIPZ. ously (10, 11). The authenticity of PC3-mm2 and C4-2B4 cell fi fi lines was con rmed by ngerprinting. pGIPZ lentiviral human Reverse transcription and quantitative PCR analysis fi PTK6 shRNA was from Thermo Scienti c. RSK1, pRSK(T359/ Total RNA was extracted from cells using the RNeasy Mini Kit S363), CKAP2, b-actin antibodies were from Santa Cruz Biotech- (Qiagen). Quantitative real-time RT-PCR (qRT-PCR) was per- nology. Anti-RSK2 antibody (clone Y83) was from Epitomics. formed, using GAPDH as a control. The PCR primer sequences Antibodies against total RSK (RSK1/RSK2/RSK3), p38-MAPK are listed in Supplementary Table S1. (D13E1), phospho p38-MAPK (Thr180/Tyr182) (D3F9), SAPK/ JNK (56G8), p-SAPK/JNK (Thr183/Tyr185) (81E11) were from Generation of C4-2B4/RSK and C4-2B4/vector cells Cell Signaling Technology. Antibodies against PTK6 and ING3 overexpressing ING3 and CKAP2 were from Proteintech. The myrRSK plasmid was kindly provided cDNAs encoding human ING3 (accession number: NM_ by Dr. John Blenis (Harvard Medical School). 019071.2) and CKAP2 (accession number: BC136332.1) were cloned by PCR and used to generate retroviral vectors. The Immunohistochemistry primer sequences used are listed in Supplementary Table S1. fi fi Formalin- xed, paraf n-embedded human prostate cancer C4-2B4/RSK and C4-2B4/vector cells infected with retroviruses specimens from primary tumor (20 cases), lymph node metastasis containing ING3 or CKAP2 cDNA were selected by 400 mg/mL (19 cases), and bone metastasis (20 cases) were obtained from G418. M.D. Anderson Cancer Center (MDACC) Prostate Cancer Tissue Bank through an institutional-approved IRB protocol. Immuno- histochemistry using pRSK(T359/S363) antibodies (Santa Cruz Biotechnology) was performed using procedures described previously (11). The staining was defined as positive when >10% of the tumor cells in the specimen were immunoreactive.

Generation of C4-2B4 cells overexpressing myrRSK cDNA encoding myrRSK (12) was inserted into bicistronic retroviral vector pBMN-I-GFP. C4-2B4/RSK cells were generated from C4-2B4-LT cells transduced with retrovirus generated from pBMN-RSK-GFP vector and selected by FACS. C4-2B4-LT cells transduced with empty vector (C4-2B4/vector) were generated similarly.

Western blotting analysis Protein concentration was determined by Coomassie Plus assay. Proteins were separated in SDS-PAGE and immunoblotted as indicated.

Cell proliferation and soft agar colony assay Cell proliferation was determined by viable cell counting. The soft agar colony assay was performed as described by Giancotti and Ruoslahti (13). Figure 1. Expression of pRSK(T359/S363) in human prostate cancer (PCa). A, Intrabone injection, bioluminescence imaging, and mCT immunohistochemistry of pRSK(T359/S363) in human prostate cancer The luciferase-expressing prostate cancer cells were injected specimens. Paraffin-embedded human prostate cancer specimens were into the right femurs of male SCID mice. Tumor growth was immunostained with anti-pRSK(T359/S363) antibody. Primary tumors monitored weekly using bioluminescence imaging (BLI) with an are mainly negative (19/20) for pRSK(T359/S363). B, lymph node metastasis IVIS Imaging System (Xenogen). Femurs and tibias were fixed in are mainly negative (18/19) for pRSK(T359/S363). C, a significant fraction of formaldehyde, and specimens were imaged on an Explore Locus bone metastasis specimens are positive (8/20, using 10% as cutoff) for pRSK(T359/S363). The pRSK(T359/S363) protein tends to localize in the RS pre-clinical in vivo scanner (GE Medical Systems). Images were nucleus (see enlargement in inset). B, bone; T, tumor. D, percentage of reconstructed and analyzed in MicroView (Parallax Innovations, prostate cancer specimens that are positive with pRSK(T359/S363). Inc.). Magnification, 200.

www.aacrjournals.org Mol Cancer Res; 13(2) February 2015 349

Yu et al.

Figure 2. Effect of myrRSK overexpression on C4-2B4 growth in bone. A, luciferase- labeled C4-2B4 cells expressing myrRSK or empty vector were selected for positivity with Tomato RFP and GFP (left). Western blotting for the expression of RSK, pRSK- T359/S363 in C4-2B4/vector and C4-2B4/RSK cells. B, no difference in C4-2B4/vector and C4-2B4/RSK cell proliferation when cultured in 10% FBS. C, effects of serum concentrations on the survival of C4- 2B4/vector and C4-2B4/RSK cells. D, anchorage-independent growth of C4-2B4/vector and C4-2B4/RSK cells. E, growth of C4-2B4/vector and C4-2B4/RSK cells in bone as measured by bioluminescence. F, quantiﬁcation of the tumor growth in bone based on the BLI. Average BLI SEM was shown. The bioluminescence signals were higher in C4-2B4/RSK tumors compared with those in C4-2B4/vector. , P < 0.05.

Knockdown PTK6 in C4-2B4/RSK cells tumors were negative with anti-pRSK(T359/S363) staining Lentivirus-expressing PTK6 shRNAs were generated as (according to 10% cutoff value; Fig. 1A). In the lymph node described above and used to infect C4-2B4/RSK cells. metastases specimens, 18 of 19 were negative (Fig. 1B). In contrast, in specimens from bone metastases, eight of 20 cases stained Results positive (Fig. 1C and Supplementary Fig. S1). The differences in pRSK(T359/S363) expression between bone metastasis and p-RSK expression is increased in human prostate cancer lymph node metastasis or primary tumors are significant (P ¼ specimens 0.003; Fig. 1D). In addition, the majority of pRSK(T359/S363) in To examine whether RSKs are activated during prostate cancer bone metastasis specimens are localized in the nucleus (Fig. 1C, progression, we performed IHC staining of paraffin-embedded inset; Supplementary Fig. S1), consistent with the reports by Zhao human prostate cancer specimens. RSK isoforms are composed of and colleagues (15) and Chen and colleagues (16) that RSKs two distinct kinase domains, both of which are activated by undergo translocation to the nucleus following growth factor phosphorylation. Dalby and colleagues (14) showed that one of stimulation. These observations suggest that RSKs are activated the mechanisms for RSK activation involves ERK-mediated phos- in a significant fraction of metastatic prostate cancer cells in bone phorylation of two adjacent conserved residues (T359 and Ser363 and may play a role in the progression of prostate cancer in bone. in RSK1) in the linker region of RSK, and S363 phosphorylation directly activates the N-terminal kinase domain. We used anti- pRSK(T359/S363) antibody to immunostain human prostate RSK expression increases anchorage-independent growth of cancer specimens that represent various stages of prostate cancer C4-2B4 cells progression from primary prostate cancer to lymph node and To examine the role of RSK in prostate cancer bone metastasis, bone metastasis. In primary prostate cancer, 19 of 20 primary we expressed myrRSK, a constitutively activated RSK (12), in

350 Mol Cancer Res; 13(2) February 2015 Molecular Cancer Research

RSK in Prostate Cancer Bone Metastasis

Figure 3. Characterization of PC3-mm2 cells with knockdown of RSK1 and/or RSK2. A, PC3-shRSK1/2 double knockdown cells stably expressed shRNAs for both RSK1 and RSK2. To generate PC3-shRSK1/2 double knockdown cells, PC3-shRSK1 cells were infected with lentivirus from pGIPZ-shRSK2. The cells were selected by using puromycin and followed with FACS for expressing both Tomato (red) and GFP (green) ﬂuorescence proteins for the expression of luciferase and shRSK2, respectively. B, Western blots of the PC3-mm2 cell lines that stably express the RSK1, RSK2, or RSK1/2 shRNAs with antibodies against RSK1, RSK2, total RSK, or pRSK-T359/S363. Vector-transfected (pLKO, pGIPZ) PC3-mm2 cells were used as controls. Cell lysates were used directly for Western blotting except for pRSK-T359/S363, in which cell lysates were ﬁrst immunoprecipitated with anti-pRSK-T359/S363 antibody followed with Western blot with antibody against total RSK. C, effects of serum concentrations on the proliferation of PC3-mm2 cells with knockdown of RSKs. D, anchorage-independent growth of PC3-mm2 cells with knockdown of RSKs.

C4-2B4 cells, which is an androgen-independent subline derived tions of FBS, i.e., 1.0% or 0.1%, the number of C4-2B4/RSK cells is from LNCaP (17). To allow for in vivo BLI, myrRSK in a bicistronic significantly higher compared with C4-2B4/vector cells (Fig. 2C). retroviral vector (pBMN-myrRSK-GFP) was transduced into C4- These observations suggest that expression of myrRSK confers a 2B4-LT cells that stably expressed luciferase and Tomato red survival advantage for C4-2B4 cells in low serum growth fluorescence protein, also introduced by bicistronic vector conditions. (pBMN-Luc-Tomato). C4-2B4-LT cells expressing both luciferase Next, we examined whether the RSK-mediated survival can lead and myrRSK (C4-2B4/RSK) were selected by FACS for cells expres- to increased anchorage-independent growth in soft agar. When sing both GFP and Tomato (Fig. 2A, left). Control C4-2B4/vector plated in soft agar, none or very few colonies, if any, were observed cells were generated by transducing C4-2B4-LT with pBMN-I-GFP in C4-2B4/vector cells (Fig. 2D), whereas a significant number of vector. Western blot analyses showed that overexpression of RSK colonies were observed in C4-2B4/RSK plates (Fig. 2D). Thus, in C4-2B4 cells not only increased the levels of total RSK but also myrRSK expression confers anchorage-independent growth of the phosphorylation of RSK at T359/S363, consistent with the C4-2B4 cells. expression of a constitutively activated RSK (Fig. 2A, right). Next, we examined the effects of RSK on C4-2B4 proliferation as Overexpression of constitutively activated RSK in C4-2B4 RSKs are known to regulate cell-cycle progression through mod- increases C4-2B4 tumor progression in bone ulation of protein factors that play a role in G1,G1–S, or G2–M One critical feature for metastatic prostate cancer cells to transition (for review, see ref. 2). In prostate cancer, studies by progress in bone is to survive in the bone microenvironment. It Clark and colleagues (8) showed that treatment of LNCaP and is possible that myrRSK-mediated anchorage-independent PC3 with the RSK inhibitor SL0101 led to inhibition of prostate growth of C4-2B4 cells may enhance C4-2B4 tumor growth in cancer cell proliferation. We found that there is no significant bone. To examine the effect of myrRSK on C4-2B4 tumor growth difference in cell proliferation between C4-2B4/RSK and the in bone, the C4-2B4/vector and C4-2B4/RSK cells were injected control C4-2B4/vector cells when the cells were cultured in media into femurs of SCID mice, and the tumor growth in bone was containing 10% (Fig. 2B) or 5% (Fig. 2C) FBS, indicating that monitored by bioluminescence. The bioluminescence signals expression of myrRSK in C4-2B4 cells does not affect their were significantly higher in C4-2B4/RSK tumors compared with proliferation under standard culture condition. However, when those in C4-2B4/vector tumors (Fig. 2E). Quantification of the these cells were cultured in media containing lower concentra- bioluminescence showed that expression of myrRSK led to more

www.aacrjournals.org Mol Cancer Res; 13(2) February 2015 351

Yu et al.

Figure 4. Effect of RSK knockdown on PC3- mm2 growth in bone. A, PC3-mm2 cells with knockdowns of RSK1, RSK2, or both RSK1/2 were injected in to mouse femurs. Control vector- transfected cells (pLKO and pGIPZ) were injected in parallel. BLI at 3 weeks after inoculation is shown. B, semiquantiﬁcation of tumor growth based on signals from BLI. Average BLI SEM was shown. Knockdown of RSK1 or both RSK1/2 resulted in signiﬁcant decreases in PC3-mm2 tumor growth in bone. , P < 0.05. C, mCT of tumor-bearing femurs. D, bone volume of tumor-bearing bone determined by mCT analysis. Bone volume within 11 mm from femur head was calculated.

than a 10-fold increase in tumor growth compared with vector- pared with pLKO or pGIPZ vector-transfected cells (Fig. 3B). To transfected cells (Fig. 2F). Together, these results indicate that determine the proportion of RSK1 or RSK2 in total RSK in PC3- increased expression of activated RSK in C4-2B4 cells enhances mm2 cells, Western blot of total RSK was performed on these RSK their growth in bone, likely through an increase in cell survival. knockdown cells. As shown in Fig. 3B, RSK1 is the major RSK in PC3-mm2 cells as knockdown of RSK1 led to a significant reduc- Knockdown of RSK1 or RSK2 expression decreases anchorage- tion (78%) of total RSK, whereas knockdown of RSK2 only led to a independent growth of PC3-mm2 cells modest (8%) decrease in total RSK. Western blotting with anti- We further determined the effect of RSK knockdown on pros- pRSK antibodies showed that knockdown of RSK also resulted in tate cancer progression in bone. Four RSK isoforms, RSK1–4, have decreases in the phosphorylation of RSK at T359/S363 (Fig. 3B). been reported in mammalian cells. Among them, RSK1 and RSK2 We further examined the effect of RSK knockdown on PC3-mm2 have been studied more extensively. Thus, we determined their cell survival. Similar to C4-2B4 cells, we found that knockdown of roles in prostate cancer growth in bone. PC3-mm2 cells that were RSKs in PC3-mm2 cells had no effect on cell proliferation when the derived from prostate cancer bone metastasis and exhibit robust cells were grown in medium with 10% serum (Fig. 3C, left). tumor growth in bone were selected for the study. Lentiviral However, knockdown of RSK1 or both RSK1/2 led to a decrease shRNAs were used to knockdown RSK1 or RSK2, individually or in cell survival when cultured in 1% serum condition (Fig. 3C, together. The lentiviral vector pLKO, with a puromycin selection right). Decreases in colony formation in soft agar were also marker, was used to knock down RSK1 and the lentivral vector observed in PC3/shRSK1 and PC3/RSK1/2 cells compared with pGIPZ, with both puromycin and GFP selection markers, was controls (Fig. 3D). The differences in the anchorage-independent used to knock down RSK2. PC3-mm2 cells with knockdown of growth of RSKs knockdown PC3-mm2 cells are not nearly as much both RSK1 and RSK2 were generated by selecting for the expres- as that observed in myrRSK-overexpressing C4-2B4 cells, suggest- sion of shRSK1 through puromycin resistance followed with ing that RSK1 is one of many factors that contribute to the transfection of pGIPZ-shRSK2 and selection for the expression anchorage-independent growth of PC3-mm2 cells. of shRSK2 through fluorescent activated cell sorting for GFP. As a result, the PC3-mm2 RSK1/2 double knockdown cells were Knockdown of RSK on PC3-mm2 tumor progression in bone resistant to puromycin (for shRSK1), and positive for GFP (for Next, we examined the effects of RSK knockdown on the growth shRSK2) and Tomato (for luciferase; Fig. 3A). Western blotting of PC3-mm2 cells in bone. PC3-mm2 cells transduced with vector using antibodies specific to RSK1 or RSK2 was used to confirm (pLKO, pGIPZ) or with single or double RSK1 and RSK2 knock- knockdown of RSK1 or RSK2 in these cell lines. As expected, the down were injected into mouse femurs. Tumor growth was levels of RSK1 or RSK2 proteins were significantly reduced com- monitored weekly by BLI, and the tumor growth at 3 weeks was

352 Mol Cancer Res; 13(2) February 2015 Molecular Cancer Research

RSK in Prostate Cancer Bone Metastasis

shown in Fig. 4A. Quantification of bioluminescence signals correspond to the changes in the levels of messages (Fig. 5B–D, demonstrated that knockdown of RSK1 in PC3-mm2 cells led to right plots). a significant inhibition of their progression in bone, whereas RSK2 knockdown did not (Fig. 4B). Knockdown of both RSK1 and RSK2 ING3 is involved in RSK-mediated C4-2B4 cell survival generated a reduction similar to knockdown of RSK1 alone (Fig. We examined whether the decrease in message levels of ING3 is 4B). These results show that RSK1 is largely responsible for RSK- involved in RSK-mediated anchorage-independent growth. ING3 mediated PC3-mm2 progression in bone, likely due to the higher is a tumor suppressor that has been shown to be involved in levels of RSK1 in PC3-mm2 cells (Fig. 3B). apoptosis and cell cycle (19). To examine whether decrease of PC3-mm2 is known to induce strong osteolytic bone lesions. ING3 expression is involved in the RSK-mediated prostate cancer Thus, we also evaluated the effects of RSK1 and RSK2 on PC3- cell survival, we overexpressed ING3 in C4-2B4/RSK cells using a mm2–induced bone changes. Analysis of tumor-bearing femurs by bicistronic retroviral vector with a neomycin selection marker. C4- X-ray showed that PC3-pLKO and PC3-pGIPZ cells induced strong 2B4/vector cells transfected with empty neomycin vector were osteolytic lesions (Supplementary Fig. S2). Non–tumor-bearing femurs do not show radiographic changes. Knockdown of RSK1 or both RSK1 and RSK2 showed a reduction of the osteolytic lesion compared with PC3 vector-injected femurs (Supplementary Fig. S2). Knockdown of RSK2 showed moderate reduction of the osteolytic response (Supplementary Fig. S2). To better define the effect of RSK knockdown on tumor-induced bone changes, the femurs were subjected to mCT analysis (Fig. 4C) and the bone volumes determined (Fig. 4D). When compared with PC3-pLKO or PC3-pGIPZ–bearing femurs, tumor-bearing femurs with PC3- shRSK1 or PC3-shRSK1/2 cells had higher bone volumes (Fig. 4D), likely due to the slower growth of PC3-shRSK1 or PC3-shRSK1/2 cells in bone. These observations suggest that knocking down RSK1 or RSK1/2 in PC3-mm2 cells led to a decrease in tumor growth in bone accompanied by a decrease in bone destruction. Together, both overexpression and knockdown approaches support a role of RSK in prostate cancer progression in bone.

Signal pathways regulating RSK-mediated survival in C4-2B4 cells We then examined signal pathways that regulate RSK-mediated events. RSKs have been shown to affect many cellular functions through multiple signal pathways. As our data above indicated RSK regulated the survival of prostate cancer cells, we focus on signal pathways that may be involved in regulating RSK-mediated survival. Jin and colleagues (18) reported that RSK2 mediates survival of head and neck cancer cells by both transcription- independent, e.g., phosphorylation of p38 MAPK and SAPK/JNK, and transcription-dependent mechanisms, e.g., expression of inhibitor of growth protein 3 (ING3), cytoskeleton-associated protein-2 (CKAP2), and protein-tyrosine kinase (PTK6/Brk). Thus, we examined whether these mechanisms are also involved in RSK-mediated survival of prostate cancer cells. The effect of myrRSK expression on p38 MAPK and SAPK/JNK phosphorylation was examined by Western blot analysis. As shown in Fig. 5A, the phosphorylation of p38 MAPK and SAPK/JNK was decreased signiﬁcantly in C4-2B4/RSK cells compared with C4-2B4/vector cells (Fig. 5A), suggesting that inhibition of these proapoptotic factors may be involved in RSK-mediated survival in prostate cancer cells. The effects of RSK expression on ING3, CKAP2, and PTK6 expression were also examined. As shown in Fig. 5B–D (left plots), expression of myrRSK in C4-2B4 cells led to decreases in the message levels of the tumor suppressors ING3 and CKAP2 and an increase in the message for PTK6, an intracellular nonreceptor tyrosine kinase, as determined by RT-PCR. qRT-PCR further Figure 5. Effects of RSK on the phosphorylation of p38-MAPK and SAPK/JNK and the showed that expression of myrRSK in C4-2B4 cells led to 65% expression of PTK6, ING3, and CKAP2. A, RSK decreases the phosphorylation and 80% decreases in ING3 and CKAP2 messages, respectively, of p38-MAPK and SAPK/JNK. V, C4-2B4/vector; RSK, C4-2B4/RSK. B to D, and a 40.0-fold increase in PTK6 message (Fig. 5B–D, middle RSK downregulates ING3, CKAP2, and upregulates PTK6 expression. Left plots). Western blot showed that the changes in the protein levels plots, RT-PCR. Middle plots, qRT-PCR. Right plots, Western blot. "", <0.05.

www.aacrjournals.org Mol Cancer Res; 13(2) February 2015 353

Yu et al.

Involvement of CKAP2 in RSK-mediated C4-2B4 cell survival CKAP2 is a cytoplasmic protein associated with cytoskeletal ﬁbers (20). CKAP2 enhances wild-type (WT) p53 activity and triggers G1 arrest and apoptosis in a p53-dependent manner (21). To examine whether CKAP2 is involved in the RSK-mediated prostate cancer cell survival, we overexpressed CKAP2 in C4-2B4/ RSK and C4-2B4/vector cells (Fig. 6D). When these cells were cultured in medium containing 0.1% FBS for three days, CKAP2 overexpression decreased C4-2B4/RSK cell numbers (Fig. 6E). Overexpression of CKAP2 in control C4-2B4/vector cells also decreased cell numbers (Fig. 6E), consistent with the function of CKAP2 in cell-cycle inhibition (21–23). Importantly, overexpression of CKAP2 inhibits RSK-mediated anchorage-independent growth in soft agar assay (Fig. 6F). These observations suggest that CKAP2 is also involved in the RSK-mediated prostate cancer survival.

Involvement of PTK6 in RSK-mediated C4-2B4 cell survival PTK6, which is upregulated by RSK activation in C4-2B4 cells (Fig. 5D), has been shown to mediate a range of cellular processes related to the development or maintenance of malignancy (24, 25). Irie and colleagues (26) reported that PTK6 regulates IGF-1– induced anchorage-independent survival of mammary epithelial cells. In addition, Harvey and colleagues (27) showed that PTK6/ Brk enhances breast carcinoma cell survival in suspension. These observations suggest that PTK6 may also play a role in RSK- mediated anchorage-independent growth. To test this, PTK6 in C4-2B4/RSK cells was knocked down by lentiviral vectors expressing PTK6 shRNAs or non-silencing (NS) shRNA. The C4-2B4/ RSK/shPTK6 cells (clones #4 and #5) and C4-2B4/RSK/shNS control cells were selected for puromycin resistance (for shRNA vectors) as well as GFP positivity (for RSK vectors). Western blotting using antibodies specific to PTK6 confirmed that C4- 2B4/RSK/shPTK6-#4 and #5 clones have significantly reduced PTK6 levels compared with shNS vector-transfected cells (Fig. 7A). In the survival assay with low serum medium, knockdown of PTK6 slightly decreased the RSK-mediated survival in C4-2B4/ RSK cells after culturing for more than five days (Fig. 7B). The Figure 6. changes in survival in low serum medium from PTK6 knockdown Effects of ING3 and CKAP2 on RSK-mediated cell survival and anchorage- are not as striking as those observed with ING3 and CKAP2 independent cell growth. A to C, overexpression of ING3 in C4-2B4/RSK cells. overexpression. However, knockdown of PTK6 significantly D to F, overexpression of CKAP2. A and D, Western blots for the expression of decreased anchorage-independent growth compared with C4- ING3 or CKAP2, respectively. B and E, cell numbers when cultured under low serum condition. C and F, anchorage-independent growth. "", <0.05. 2B4/RSK-shNS control cells (Fig. 7C). On the basis of these observations, we propose that RSK-mediated transcriptional regulation of ING3, CKAP2, and PTK6 plays a role in RSK-mediated C4-2B4 cell survival in vitro, and possibly C4-2B4 progression in used as a control. The C4-2B4/RSK/ING3 and control cells were bone in vivo (Fig. 7D). selected for neomycin resistance (for ING3 vector) as well as GFP positivity (for myrRSK vector). As shown in Fig. 6A, expression of ING3 in C4-2B4/RSK cells restored ING3 to a slightly higher level Discussion than that in control C4-2B4/vector cells. C4-2B4/vector cells Survival in the bone marrow microenvironment is one of overexpressing ING3 (C4-2B4/vector/ING3) were also generated the key steps for the metastatic growth of prostate cancer to test the effect of ING3 on C4-2B4 cells. When these cells were cells in bone. Our studies showed that RSK increases pros- cultured in medium containing low serum, i.e., 1% FBS, over- tate cancer cell survival in vitro and progression in bone in expression of ING3 decreased the cell number when cultured for vivo. In addition, we showed that RSK phosphorylation is more than four days (Fig. 6B). Expression of ING3 also abrogated increased in human prostate cancer bone metastasis speci- RSK-mediated anchorage-independent growth in C4-2B4/RSK mens compared with those in primary site or lymph node cells (Fig. 6C). Overexpression of ING3 in C4-2B4/vector cells metastasis. Together, our studies provide evidence that RSK did not have significant effects on either assay (Fig. 6B and C). is an important driver for the progression of prostate cancer These results suggest that ING3 plays a role in RSK-mediated C4- in bone and suggest that RSK is a promising therapy target 2B4 cell survival. forprostatecancerbonemetastasis.

354 Mol Cancer Res; 13(2) February 2015 Molecular Cancer Research

RSK in Prostate Cancer Bone Metastasis

Figure 7. Effects of PTK6 on RSK-mediated cell survival and anchorage-independent cell growth. A, Western blot for the expression of PTK6. B, cell numbers when cultured under low serum condition. C, knockdown of PTK6 attenuates RSK-mediated anchorage- independent growth. D, proposed model of RSK-mediated C4-2B4 cell survival and progression in bone. Increase in RSK activity leads to transcriptional downregulation of ING3 and CKAP2 and upregulation of PTK6 in C4-2B4 cells. These lead to increased survival of C4-2B4 cells in vitro and progression in bone in vivo."", <0.05.

RSKs mediate different cellular functions in a context-dependent RSK has been shown to exhibit transcriptional regulation of manner (1). RSKs are known to regulate cell-cycle progression (for gene expression. Indeed, we showed that in human prostate review, see ref. 2). Studies by Clark and colleagues (8) showed that cancer bone metastasis specimens, phosphorylated RSK is mainly RSK inhibition decreases the proliferation of cancer cells, including localized in the nucleus, suggesting a possible role in gene LNCaP and PC3 prostate cancer cells and MCF-7 breast cancer cells, regulation. Studies by Chen and colleagues (16) showed that but not normal breast epithelial cells MCF-10A (8), suggesting that nuclear RSK plays a role in modulating c-Fos phosphorylation. We cancer cells may exploit RSK-mediated pathways for proliferation. found that RSKs transcriptionally regulated the expression of Although Clark and colleagues (8) showed that treatment of several apoptosis-related genes, including PTK6, ING3, and LNCaP and PC3 cells with RSK inhibitor SL0101 led to inhibition CKAP2 (18), in C4-2B4 prostate cancer cell line. As PTK6, ING3, of prostate cancer cell proliferation, we found that overexpression and CKAP2 are CREB target genes (18), RSK may regulate these of myrRSK in C4-2B4 or knockdown of RSK1 in PC3-mm2 cells genes through CREB phosphorylation. Interestingly, the functions mainly affected anchorage-independent growth, but not cell pro- of ING3 and CKAP2 are p53 dependent, and C4-2B4 has WT p53 liferation, when the cells were cultured under standard culture (28). We found that knockdown of RSK1/2 in PC3-mm2 cells did condition. The differences between our studies and those of Clark not affect the expression of ING3 or CKAP2 (data not shown), and colleagues (8) may be due to the difference in cell lines, i.e., likely due to the lack of functional p53 in PC3-mm2 cells (28). LNCaP versus C4-2B4, and the approach, i.e., inhibitor versus gene How RSK regulates survival of PC3-mm2 cells is not known and expression, used in these studies. requires further analysis.

www.aacrjournals.org Mol Cancer Res; 13(2) February 2015 355

Yu et al.

Our observation that RSKs play a role in prostate cancer bone upregulating MMP2, without affecting tumor growth in bone. In metastasis suggests that RSKs have potential to be a therapy target this study, we found that knockdown of RSK1 in PC3-mm2 cells for prostate cancer bone metastasis. As Clark and colleagues (8) leads to a decrease in cell survival, resulting in a decrease of showed that RSK inhibition preferentially inhibits the prolifera- osteolytic response. Thus, therapies targeting RSK may also help tion of both androgen-dependent LNCaP and androgen-indepen- in reducing tumor-associated bone lesions. dent PC3 prostate cancer cells, but not normal breast epithelial In conclusion, our studies identify a novel role of RSK in cells, these interesting observations also suggest that it is possible prostate cancer bone metastasis through both transcriptional and to preferentially inhibit RSK activity in cancer cells. RSK has been posttranslational modulation of pathways that regulate anchor- shown to be a druggable target. Three different classes of RSK age-independent growth. Further development of strategies that inhibitors, SL0101 (29), FMK (30) and BI-D1870 (31), have been inhibit RSK-mediated survival in prostate cancer is warranted. identified (32). However, a relatively high concentration, i.e., in mmol/L range, is required for these inhibitors to elicit an effect on Disclosure of Potential Conflicts of Interest cellular activity in vitro. Clark and colleagues (8) showed that No potential conflicts of interest were disclosed. SL0101 at a concentration of 20 mmol/L inhibited LNCaP or PC3 proliferation in vitro. Jin and colleagues (18) showed anoikis Authors' Contributions sensitization of several cancer cell lines by using SL0101 and Conception and design: G. Yu, Y.-C. Lee, J.H. Song, G.E. Gallick, L.-Y. Yu-Lee, FMK at the concentrations of 100 mmol/L and 10 mmol/L, respec- J. Kuang, S.-H. Lin Development of methodology: G. Yu, Y.-C. Lee, C.-F. Wu, J.H. Song, J. Kuang tively. Similarly, a high concentration of BI-D1870 (10 mmol/L) Acquisition of data (provided animals, acquired and managed patients, was also required for detecting its effect in cell lines (31). In provided facilities, etc.): Y.-C. Lee, C.-J. Cheng, C.-F. Wu, J.H. Song, G.E. Gallick addition, BI-D1870 also has effects on polo-kinase 1 activity (31), Analysis and interpretation of data (e.g., statistical analysis, biostatistics, leading to failure of cells to undergo cytokinesis in BI-D1870– computational analysis): G. Yu, Y.-C. Lee, C.-J. Cheng, C.-F. Wu, J.H. Song, treated C4-2B4 cells (data not shown). Development of RSK G.E. Gallick, L.-Y. Yu-Lee, J. Kuang, S.-H. Lin inhibitors with improved affinity, biologic stability, and mem- Writing, review, and/or revision of the manuscript: Y.-C. Lee, C.-J. Cheng, J.H. Song, G.E. Gallick, L.-Y. Yu-Lee, J. Kuang, S.-H. Lin brane permeability of SL0101 has been reported (33, 34). Thus, it Administrative, technical, or material support (i.e., reporting or organizing is likely that a clinically applicable inhibitor for RSK will be data, constructing databases): G. Yu, Y.-C. Lee, S.-H. Lin available in the near future. Alternatively, it is possible to target Study supervision: S.-H. Lin downstream mediators of RSK. Further development of inhibitors of RSK activity or RSK-mediated signal pathways will have poten- Grant Support tial in the treatment of prostate cancer bone metastasis. This work was supported by grants from the NIH CA174798, P50 CA140388, Metastatic prostate cancer cells in bone may affect bone homeo- CA16672, the Prostate Cancer Foundation, DOD (PC093132), DOD stasis, resulting in osteoblastic or osteolytic responses. PC3 cells (PC080847), and the Cancer Prevention and Research Institute of Texas (CPRIT have been shown to generate osteolytic bone lesions. Tumor cells RP110327). The costs of publication of this article were defrayed in part by the can modulate osteolytic program through various mechanisms. payment of page charges. This article must therefore be hereby marked Hall and colleagues (35) showed that knockdown of DKK-1 in advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate PC3 cells inhibited cell proliferation, through induction of this fact. p21CIP-1/WAF-1, resulting in a reduction of osteolytic lesion in bone. In contrast, Dutta and colleagues (36) showed that integrin Received July 10, 2014; revised August 20, 2014; accepted August 24, 2014; avb6 expression promotes an osteolytic program in PC3 cells by published OnlineFirst September 4, 2014.

References 1. Sulzmaier FJ, Ramos JW. RSK isoforms in cancer cell invasion and metas- 10. Huang CF, Lira C, Chu K, Bilen MA, Lee YC, Ye X, et al. Cadherin-11 tasis. Cancer Res 2013;73:6099–105. increases migration and invasion of prostate cancer cells and enhances their 2. Romeo Y, Zhang X, Roux PP. Regulation and function of the RSK family of interaction with osteoblasts. Cancer Res 2010;70:4580–9. protein kinases. Biochem J 2012;441:553–69. 11. Chu K, Cheng CJ, Ye X, Lee YC, Zurita AJ, Chen DT, et al. Cadherin-11 3. Lara R, Seckl MJ, Pardo OE. The p90 RSK family members: common promotes the metastasis of prostate cancer cells to bone. Mol Cancer Res functions and isoform specificity. Cancer Res 2013;73:5301–8. 2008;6:1259–67. 4. Anjum R, Blenis J. The RSK family of kinases: emerging roles in cellular 12. Richards SA, Dreisbach VC, Murphy LO, Blenis J. Characterization of signalling. Nat Rev Mol Cell Biol 2008;9:747–58. regulatory events associated with membrane targeting of p90 ribosomal 5. Bjorbaek C, Zhao Y, Moller DE. Divergent functional roles for p90rsk kinase S6 kinase 1. Mol Cell Biol 2001;21:7470–80. domains. J Biol Chem 1995;270:18848–52. 13. Giancotti FG, Ruoslahti E. Elevated levels of the alpha 5 beta 1 fibronectin 6. Wang R, Jung SY, Wu CF, Qin J, Kobayashi R, Gallick GE, et al. Direct roles receptor suppress the transformed phenotype of Chinese hamster ovary of the signaling kinase RSK2 in Cdc25C activation during Xenopus oocyte cells. Cell 1990;60:849–59. maturation. Proc Natl Acad Sci U S A 2010;107:19885–90. 14. Dalby KN, Morrice N, Caudwell FB, Avruch J, Cohen P. Identification of 7. Wu CF, Liu S, Lee YC, Wang R, Sun S, Yin F, et al. RSK promotes G2/M regulatory phosphorylation sites in mitogen-activated protein kinase transition through activating phosphorylation of Cdc25A and Cdc25B. (MAPK)-activated protein kinase-1a/p90rsk that are inducible by MAPK. Oncogene 2014;33:2385–94. J Biol Chem 1998;273:1496–505. 8. Clark DE, Errington TM, Smith JA, Frierson HF Jr., Weber MJ, Lannigan DA. 15. Zhao Y, Bjorbaek C, Weremowicz S, Morton CC, Moller DE. RSK3 encodes The serine/threonine protein kinase, p90 ribosomal S6 kinase, is an a novel pp90rsk isoform with a unique N-terminal sequence: growth important regulator of prostate cancer cell proliferation. Cancer Res factor-stimulated kinase function and nuclear translocation. Mol Cell Biol 2005;65:3108–16. 1995;15:4353–63. 9. Zhang X, Lavoie G, Fort L, Huttlin EL, Tcherkezian J, Galan JA, et al. Gab2 16. Chen RH, Abate C, Blenis J. Phosphorylation of the c-Fos transrepression phosphorylation by RSK inhibits Shp2 recruitment and cell motility. Mol domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 Cell Biol 2013;33:1657–70. kinase. Proc Natl Acad Sci U S A 1993;90:10952–6.

356 Mol Cancer Res; 13(2) February 2015 Molecular Cancer Research

RSK in Prostate Cancer Bone Metastasis

17. Thalmann GN, Sikes RA, Wu TT, Degeorges A, Chang SM, Ozen M, et al. 27. Harvey AJ, Pennington CJ, Porter S, Burmi RS, Edwards DR, Court W, et al. LNCaP progression model of human prostate cancer: androgen-indepen- Brk protects breast cancer cells from autophagic cell death induced by loss dence and osseous metastasis. Prostate 2000;44:91–103. of anchorage. Am J Pathol 2009;175:1226–34. 18. Jin L, Li D, Lee JS, Elf S, Alesi GN, Fan J, et al. p90 RSK2 mediates antianoikis 28. Carroll AG, Voeller HJ, Sugars L, Gelmann EP. p53 oncogene mutations in signals by both transcription-dependent and -independent mechanisms. three human prostate cancer cell lines. Prostate 1993;23:123–34. Mol Cell Biol 2013;33:2574–85. 29. Smith JA, Poteet-Smith CE, Xu Y, Errington TM, Hecht SM, Lannigan DA. 19. Nagashima M, Shiseki M, Pedeux RM, Okamura S, Kitahama-Shiseki M, Identification of the first specific inhibitor of p90 ribosomal S6 kinase Miura K, et al. A novel PHD-finger motif protein, p47ING3, modulates (RSK) reveals an unexpected role for RSK in cancer cell proliferation. p53-mediated transcription, cell cycle control, and apoptosis. Oncogene Cancer Res 2005;65:1027–34. 2003;22:343–50. 30. Cohen MS, Zhang C, Shokat KM, Taunton J. Structural bioinformatics- 20. Maouche-Chretien L, Deleu N, Badoual C, Fraissignes P, Berger R, Gaulard based design of selective, irreversible kinase inhibitors. Science 2005;308: P, et al. Identification of a novel cDNA, encoding a cytoskeletal associated 1318–21. protein, differentially expressed in diffuse large B cell lymphomas. Onco- 31. Sapkota GP, Cummings L, Newell FS, Armstrong C, Bain J, Frodin M, et al. gene 1998;17:1245–51. BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) 21. Tsuchihara K, Lapin V, Bakal C, Okada H, Brown L, Hirota-Tsuchihara M, isoforms in vitro and in vivo. Biochem J 2007;401:29–38. et al. Ckap2 regulates aneuploidy, cell cycling, and cell death in a p53- 32. Nguyen TL. Targeting RSK: an overview of small molecule inhibitors. dependent manner. Cancer Res 2005;65:6685–91. Anticancer Agents Med Chem 2008;8:710–6. 22. Seki A, Fang G. CKAP2 is a spindle-associated protein degraded by APC/C- 33. Mrozowski RM, Vemula R, Wu B, Zhang Q, Schroeder BR, Hilinski MK, Cdh1 during mitotic exit. J Biol Chem 2007;282:15103–13. et al. Improving the affinity of SL0101 for RSK using structure-based design. 23. Hong KU, Park YS, Seong YS, Kang D, Bae CD, Park J. Functional impor- ACS Med Chem Lett 2012;4:175–9. tance of the anaphase-promoting complex-Cdh1-mediated degradation of 34. Hilinski MK, Mrozowski RM, Clark DE, Lannigan DA. Analogs of the RSK TMAP/CKAP2 in regulation of spindle function and cytokinesis. Mol Cell inhibitor SL0101: optimization of in vitro biological stability. Bioorg Med Biol 2007;27:3667–81. Chem Lett 2012;22:3244–7. 24. Ostrander JH, Daniel AR, Lange CA. Brk/PTK6 signaling in normal and 35. Hall CL, Zhang H, Baile S, Ljungman M, Kuhstoss S, Keller ET. p21CIP-1/ cancer cell models. Curr Opin Pharmacol 2010;10:662–9. WAF-1 induction is required to inhibit prostate cancer growth elicited by 25. Brauer PM, Tyner AL. Building a better understanding of the intracellular deficient expression of the Wnt inhibitor Dickkopf-1. Cancer Res tyrosine kinase PTK6 - BRK by BRK. Biochim Biophys Acta 2010;1806: 2010;70:9916–26. 66–73. 36. Dutta A, Li J, Lu H, Akech J, Pratap J, Wang T, et al. Integrin alphavbeta6 26. Irie HY, Shrestha Y, Selfors LM, Frye F, Iida N, Wang Z, et al. PTK6 regulates promotes an osteolytic program in cancer cells by upregulating MMP2. IGF-1-induced anchorage-independent survival. PLoS One 2010;5:e11729. Cancer Res 2014;74:1598–608.

www.aacrjournals.org Mol Cancer Res; 13(2) February 2015 357

RSK Promotes Prostate Cancer Progression in Bone through ING3, CKAP2, and PTK6-Mediated Cell Survival

Guoyu Yu, Yu-Chen Lee, Chien-Jui Cheng, et al.

Mol Cancer Res 2015;13:348-357. Published OnlineFirst September 4, 2014.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-14-0384-T

Supplementary Access the most recent supplemental material at: Material http://mcr.aacrjournals.org/content/suppl/2014/09/05/1541-7786.MCR-14-0384-T.DC1

Cited articles This article cites 36 articles, 22 of which you can access for free at: http://mcr.aacrjournals.org/content/13/2/348.full#ref-list-1

Citing articles This article has been cited by 4 HighWire-hosted articles. Access the articles at: http://mcr.aacrjournals.org/content/13/2/348.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mcr.aacrjournals.org/content/13/2/348. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.