Activated α₂-Macroglobulin Regulates Transcriptional Activation of c-MYC Target Genes through Cell Surface GRP78 Protein

Abstract

Activated α₂-macroglobulin (α₂M*) signals predominantly through cell surface GRP78 (CS-GRP78) to promote proliferation and survival of cancer cells; however, the molecular mechanism remains obscure. c-MYC is an essential transcriptional regulator that controls cell proliferation. We hypothesize that α₂M*/CS-GRP78-evoked key signaling events are required for transcriptional activation of c-MYC target genes. Activation of CS-GRP78 by α₂M* requires ligation of the GRP78 primary amino acid sequence (Leu⁹⁸–Leu¹¹⁵). After stimulation with α₂M*, CS-GRP78 signaling activates 3-phosphoinositide-dependent protein kinase-1 (PDK1) to induce phosphorylation of PLK1, which in turn induces c-MYC transcription. We demonstrate that PLK1 binds directly to c-MYC and promotes its transcriptional activity by phosphorylating Ser⁶². Moreover, activated c-MYC is recruited to the E-boxes of target genes FOSL1 and ID2 by phosphorylating histone H3 at Ser¹⁰. In addition, targeting the carboxyl-terminal domain of CS-GRP78 with a mAb suppresses transcriptional activation of c-MYC target genes and impairs cell proliferation. This work demonstrates that α₂M*/CS-GRP78 acts as an upstream regulator of the PDK1/PLK1 signaling axis to modulate c-MYC transcription and its target genes, suggesting a therapeutic strategy for targeting c-MYC-associated malignant progression.

Introduction

α₂-Macroglobulin (α₂M)² is a plasma protein that interacts with and entraps virtually all proteinases, thereby blocking access to their substrates (1). In prostate cancer patients, α₂M is proteolytically activated (α₂M*) and signals predominantly through interaction with cell surface GRP78 (K_d ∼ 50–100 pm), promoting proliferation and survival of cancer cells (2, 3). GRP78 is a stress-inducible, prosurvival, endoplasmic reticulum chaperone belonging to the HSP70 family. It is composed of an ATPase domain, a peptide binding domain, and a COOH-terminal domain of unknown function (4–6). Several different cell types, including proliferating endothelial cells and tumor cells, express GRP78 on their surface (7–15). GRP78 expression at the cell surface and its ligation by α₂M* are clearly implicated in the development of metastatic prostate cancer (2, 9, 16–19). However, the mechanism by which α₂M*/cell surface GRP78 (CS-GRP78) signaling regulates gene transcription and their responses in cell proliferation is unknown.

CS-GRP78 is a multifunctional receptor that forms complexes with phosphatidylinositol 3-kinase (PI3K) and enhances phosphatidylinositol 3,4,5-trisphosphate production, consistent with its novel role as a regulator of the PI3K/Akt signaling pathway. Thus it promotes cell proliferation, survival, metastasis, and chemoresistance (9, 20–22). CS-GRP78, through its NH₂-terminal domain, drives PI3K/Akt activity (2), whereas targeting the COOH-terminal domain with antibody promotes apoptotic signaling (21, 23). Recently, we demonstrated in vivo that targeting the GRP78 COOH-terminal domain with monoclonal antibody C38 (C38 mAb) delays tumor growth and prolongs survival (15). We also demonstrated that α₂M*/CS-GRP78 signaling is required for mechanistic target of rapamycin (mTOR) complex-mediated phosphorylation of Akt by 3-phosphoinositide-dependent protein kinase-1 (PDK1) (22). PDK1 regulates a diversity of substrates and targets that induce aberrant signaling in human malignancy (24). Indeed, recent studies show that PDK1 is required for c-MYC accumulation, and it regulates c-MYC activity through the downstream target PLK1 (25), indicating a potential functional link of α₂M*/CS-GRP78 signaling and c-MYC in proliferation of cancer cells. α₂M*/CS-GRP78-mediated PI3K/Akt signaling is well documented; however, its role in cancer-associated gene regulation by transcription factors has yet to be identified.

The oncogene c-MYC globally reprograms cells and drives proliferation by regulating an estimated 15% of the genes in the human genome (26). Recent work suggests that rather than acting as a general amplifier of transcription (27, 28) c-MYC activates and represses transcription of discrete gene sets, leading to changes in cell proliferation, tumor progression, and maintenance (29). Moreover, phosphorylation of c-MYC at certain sites governs its activation and subsequent biological functions through transcriptional activation of target genes that are necessary for cell proliferation. Specifically, Ser⁶² phosphorylation is necessary for its oncogenic activity (30). A key question is whether α₂M*/CS-GRP78 signaling is required for activation of c-MYC and its downstream target genes.

In the present study, we demonstrate that α₂M*/CS-GRP78-mediated PDK1/PLK1 signaling contributes to the transcriptional activation of c-MYC target genes and proliferation. We further demonstrate that PLK1 can directly bind to c-MYC and promote its transcriptional activity by phosphorylating at histone H3 Ser¹⁰ (H3S10). These findings suggest that α₂M*/CS-GRP78 signaling drives c-MYC target gene expression in human cancers and provide a therapeutic approach for targeting c-MYC-driven tumors.

Experimental Procedures

Cell Culture

1-LN prostate cancer cells were a kind gift from Dr. Philip Walther, Department of Surgery, Duke University Medical Center. They now reside in our laboratory and are available on request. DU145 prostate cancer cells, A375 melanoma cells, and U373 glioma cells were purchased from the Duke Cell Culture Facility. 1-LN and DU145 cells were maintained in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum (FBS), 1% penicillin/streptomycin at 37 °C in a 5% CO₂-humidified atmosphere. A375 and U373 cells were maintained in DMEM (high glucose; Gibco, Life Technologies) containing 10% FBS, 1% penicillin/streptomycin at 37 °C in a 5% CO₂-humidified atmosphere.

Antibodies and Reagents

Antibodies recognizing c-MYC, P-c-MYC (Ser⁶²), PDK1, P-PDK1 (Ser²⁴¹), PLK1, P-PLK1 (Thr²¹⁰), P-histone H3 (Ser¹⁰), histone H3, cleaved poly(ADP-ribose) polymerase (Asp²¹⁴), kinase buffer (10×), ATP, and SignalSilence c-MYC siRNAII were purchased from Cell Signaling Technology. GAPDH antibody was purchased from Genscript. c-MYC recombinant protein was purchased from Novus Biologicals. Cell proliferation kit II (XTT) was purchased from Roche Applied Science. Glycogen, Lipofectamine 2000, and secondary antibodies conjugated with Alexa Fluor 680, Alexa Fluor 790, and Alexa Fluor 647 were purchased from Invitrogen. IRDye 800 CW was purchased from Rockland. Protein G-agarose beads and Proteinase K were purchased from Sigma. Protein A/G magnetic beads were purchased from Thermo Scientific. BX795 and BI2536 were purchased from Selleck Chemicals. α₂M* was prepared as described previously (31). GRP78 murine monoclonal antibody (C38) was produced in our laboratory (32).

Peptides

GRP78 wild type (WT) peptide CLIGRTWNDPSVQQDIKFL (Leu⁹⁸–Leu¹¹⁵), mutant peptide LIGRTWNDPSVQQDIVFL (K¹¹³–V), and scrambled peptide GTNKSQDLWIPQLRDVFI were purchased from Genemed Synthesis, Inc.

Small Interfering RNA (siRNA) Interference and Lentiviral Transfections

siRNAs targeting c-MYC and nonspecific siRNA were transfected into 1-LN cells with Lipofectamine 2000 reagent according to the manufacturer's instructions. sh-c-MYC lentiviral particles (Clone ID TRCN000000-39642) were obtained from Sigma and transfected into 1-LN cells according to the manufacturer's instructions. After transfection with sh-c-MYC vector, 1-LN cells were selected with 2 mg/ml puromycin.

Flow Cytometry

CS-GRP78 was analyzed by flow cytometry as described previously (15). The mean fluorescence intensity of the signal was calculated by Flow Jo® software, and signal obtained from GRP78 was normalized with that obtained from isotype controls.

Cell Proliferation Assays

Cells were plated in 96-well plates at 10,000 cells/well in 0.1 ml of growth medium containing inhibitor BX795, BI2536, or C38 mAb for 72 h. Cell viability was measured according to the manufacturer's instruction protocol by using the XTT assay. Absorbance was read at λ = 450 nm.

Soft Agar Colony Formation Assay

Soft agar assays were conducted in 6-well plates in triplicate. For each well, 10,000 cells were mixed in growth medium containing 1.2% agarose and inhibitor BX795, BI2536, or C38 mAb. Cells were then layered over 2% agarose in regular medium. Medium containing BX795, BI2536, or C38 mAb was added to each well every 3 days. The assays were terminated after 21 days, colonies were counted under a microscope or stained with crystal violet, and pictures were taken.

Immunoblotting and Immunoprecipitation

Protein extracts, immunoblotting, and immunoprecipitate analysis were performed as described previously (27), and all blots are representative of a minimum of two independent experiments.

Immunoprecipitation and in Vitro Kinase Assays

PLK1 immunoprecipitation-kinase assays were performed as described previously (27) and analyzed by immunoblotting using P-c-MYC (Ser⁶²) and c-MYC antibodies.

Quantitative Real Time PCR and PCR Array

Total RNA was prepared from cells using the Quick-RNA Mini Prep kit (Zymo Research), and cDNAs were generated using the iScript cDNA synthesis kit (Bio-Rad). SYBR Green reactions were done using a Bio-Rad CFX96 quantitative real time PCR system. For data analysis, raw counts were normalized to the housekeeping gene averaged for the same time point and condition (ΔC_t). Counts are reported as -fold change relative to the untreated control (2^−ΔΔCt). All primers were designed and synthesized by Integrated DNA Technologies or Eurofins MWG Operon. Primers are listed in Table 1. Array samples were prepared according to the manufacturer's instructions by using RNeasy Plus Mini kits (Qiagen). Samples were analyzed using a human MYC target gene PCR array (Qiagen PAHS-177ZA). Genes were considered differentially expressed if they demonstrated a significant p value ≤0.05 and were at least 2-fold or greater up-regulated. An average of biological triplicates was used to generate heat maps by using Qiagen data analysis center software.

TABLE 1.

Primers used for quantitative RT-PCR analysis

Gene	Primer	Sequence
FOSL1	Forward	5′-CAG GCG GAG ACT GAC AAA CTG-3′
	Reverse	5′-TCC TTC CGG GAT TTT GCA GAT-3′
ID2	Forward	5′-AGT CCC GTG AGG TCC GTT AG-3′
	Reverse	5′-AGT CGT TCA TGT TGT ATA GCA GG-3′
c-MYC	Forward	5′-GGC TCC TGG CAA AAG GTC A-3′
	Reverse	5′-CTG CGT AGT TGT GCT GAT GT-3′

Chromatin Immunoprecipitation (ChIP) Assay

ChIP assays were done in 1-LN prostate cancer cells as described previously (33). ChIP enrichment for a primer set was evaluated by quantitative PCR as percentage of input and normalized to a negative primer set. FOSL1 and ID2 gene probes used for ChIP analysis were designed as described previously (33) and are listed in Table 2.

TABLE 2.

Primers used for ChIP-PCR assays

Gene	Primer	Sequence
FOSL1	Forward	5′-GAG ATC GAG CCA CTG CAC TC-3′
	Reverse	5′-CAA ACC AGG GTT ACA AAA AAT GG-3′
FOSL1 negative region primers	Forward	5′-CCA GAG ACA GGT TT CCC AGA-3′
	Reverse	5′-GAC CAG GCT GAA GGA AGG A-3′
ID2	Forward	5′-AAG CCC ACA CTA AGC CTG TC-3′
	Reverse	5′-GTC ACG TTT GTA GAG TGA GTT CG-3′
ID2 negative region primers	Forward	5′-CGC GCT TGG AAT TAG AAT TAA G-3′
	Reverse	5′-AAG ATG GGG GAG GGG AAG-3′

Statistical Analysis

Data are presented as mean ± S.D. unless otherwise stated. A Student's t test was used to compare two groups for statistical significance analysis. p values ≤0.05 were considered as significant.

Results

α₂M*-induced c-MYC Expression Promotes Tumor Growth in Soft Agar

We first determined the ability of α₂M* to promote proliferation by using an in vitro assay that measures anchorage-independent growth in soft agar. This assay demonstrates that α₂M* promotes a significant increase in colony formation in a variety of cancer cell lines (Fig. 1A). Furthermore, we analyzed the effect of α₂M* on cell proliferation in standard cell culture of the same panel of cancer cell lines. Consistent with our previous report, α₂M* significantly increased cell proliferation in these lines (Fig. 1B) (9). This result confirms the potential role of α₂M* in cancer cell proliferation.

FIGURE 1.

α₂M* potentiates cell proliferation through c-MYC induction. A, soft agar growth of various cancer cell lines stimulated with α₂M* (100 pm). B, cell proliferation assay showing various cancer cell lines stimulated with α₂M* (100 pm). C, immunoblotting analysis showing c-MYC expression level in 1-LN prostate cancer cell line after stimulation with α₂M* at the indicated doses and times. D, immunoblotting analysis of the indicated proteins in 1-LN cells treated with cycloheximide (CHX; 100 μg/ml) or actinomycin D (ActD; 5 μg/ml) at the indicated time points. E, c-MYC transcript expression in 1-LN prostate cancer cell line after stimulation with α₂M* at the indicated doses and time points. F, immunoblotting analysis of the indicated proteins in 1-LN cells transfected with Scr-siRNA and si-c-MYC. G, quantitative c-MYC transcript levels in α₂M*-stimulated Scr-siRNA and si-c-MYC 1-LN cells. H, Scr-shRNA and sh-c-MYC 1-LN cells stimulated with α₂M* (100 pm). I, cell proliferation assays in 1-LN cells transfected with Scr-siRNA and si-c-MYC and then stimulated with *α₂M (100 pm) for 30 min. *, p values ≤0.05. Error bars represent S.D. UT, untreated.

To determine the mechanism by which α₂M* is involved in cell proliferation, we examined the expression of c-MYC. α₂M* stimulation of 1-LN cells induces c-MYC protein expression in a dose- and time-dependent manner (Fig. 1C). Maximal c-MYC expression occurred with 100 pm α₂M* at 30 min. Of interest, the maximal effective dose of 100 pm is consistent with many previous studies, demonstrating that the maximal proliferative effect of α₂M* on proliferation occurs at this dose with a falloff in activity at 500 pm (for example, see Refs. 2 and 31). The reason for this dose effect is unknown; however, we used these conditions for subsequent experiments. Next we investigated whether α₂M* induces c-MYC at a post-translational or transcriptional level. 1-LN cells were treated with either cycloheximide or actinomycin D and then stimulated with α₂M*. α₂M* with either cycloheximide or actinomycin D decreased the c-MYC half-life, indicating that α₂M* induces c-MYC at the transcript level (Fig. 1D). α₂M* induced c-MYC transcription in a dose- and time-dependent manner (Fig. 1E). At 500 pm α₂M*, c-MYC transcription continued to increase unlike the protein expression level. Recent reports indicate that c-MYC mRNA is targeted by several microRNAs to control its expression levels (34, 35). Although actinomycin D decreased the α₂M*-induced change in c-MYC half-life, it is possible that α₂M* can induce the transcription of essential translational machineries or microRNAs required to modulate c-MYC translation.

To further substantiate the role of c-MYC activation by α₂M*, we suppressed c-MYC expression in 1-LN cells (Fig. 1F). α₂M* did not induce c-MYC transcript in these c-MYC silenced cells (Fig. 1G). Next we investigated the effect of α₂M* in Scr-sh-RNA and sh-c-MYC 1-LN cells by a soft agar assay. As expected, sh-c-MYC cells showed inhibition of colony formation, and α₂M* had no further effect (Fig. 1H). Indeed, RNA interference-mediated knockdown of c-MYC in 1-LN cells resulted in decreased proliferation, whereas α₂M* had no further effect (Fig. 1I). These data suggest that α₂M*-induced proliferation depends on c-MYC expression.

α₂M* Signals through CS-GRP78 to Induce c-MYC Expression in a PDK1/PLK1 Signaling-dependent Manner

Our previous studies demonstrate that α₂M* induces signaling pathways through its receptor CS-GRP78 promoting cancer survival and metastasis (3, 22, 23). Recently, we produced a highly specific C38 mAb directed against the COOH-terminal domain of GRP78 that shows therapeutic efficacy in reducing tumor growth in vivo (15, 32). We screened CS-GRP78 in multiple cancer cell lines (Fig. 2A). To investigate whether α₂M* induces c-MYC expression through its receptor CS-GRP78, we treated the 1-LN cells with 50 μg of C38 mAb for 6 h and then stimulated with α₂M*. We observed that targeting CS-GRP78 abrogated α₂M*-induced c-MYC transcription (Fig. 2B). Moreover, in a panel of cancer cell lines, C38 mAb treatment blocked α₂M*-mediated c-MYC protein expression (Fig. 2C, first lane). These findings demonstrate that α₂M* signaling through CS-GRP78 induces c-MYC expression.

FIGURE 2.

α₂M* ligation of CS-GRP78 induces c-MYC activation, and this requires PDK1/PLK1 signaling. A, surface expression of GRP78 was detected in the indicated cancer cell lines by flow cytometric analysis of nonpermeabilized cells. Surface GRP78 was visualized with murine monoclonal antibody C38 followed by fluorescently labeled secondary antibody, and results are reported relative to the matched isotype control. Positively stained cells are represented as the area under the respective histogram, and mean fluorescence intensity values are shown. B, 1-LN cells treated with C38 mAb (50 μg) or control IgG (50 μg) for 6 h and then stimulated with α₂M* (100 pm) for 30 min to quantify the c-MYC transcript level. C, immunoblotting analysis of the indicated cancer cell lines treated with C38 mAb (50 μg) for 6 h and then stimulated with α₂M* (100 pm) for 30 min. D, various cancer cell lines were stimulated with α₂M* (100 pm) for 30 min in the absence or presence of Scr peptide (100 pm for 1 h), GRP78 WT peptide (100 pm for 1 h), or GRP78 mutant peptide (100 pm for 1 h) and then immunoblotted for the indicated proteins. Error bars represent S.D. UT, untreated.

To investigate the downstream kinases of CS-GRP78 action crucial for c-MYC induction, we investigated PDK1/PLK1 signaling, which induces MYC phosphorylation and protein accumulation (25, 36). α₂M* induced strong phosphorylation of PDK1 (Ser²⁴¹) and increased phosphorylated PLK1 (Thr²¹⁰) in a dose- and time-dependent manner (data not shown). Further analyses in multiple cancer cell lines showed that targeting CS-GRP78 with C38 mAb abolished α₂M*-induced phosphorylation of PDK1 (Ser²⁴¹) and PLK1 (Thr²¹⁰) (Fig. 2C, third and fifth lanes). These data confirm that GRP78 is a functional molecular target on cancer cell surfaces.

In our previous studies, we identified the GRP78 primary amino acid sequence LIGRTWNDPSVQQDIKFL (Leu⁹⁸–Leu¹¹⁵) as the putative binding site for α₂M* (9). We next demonstrated the specificity of CS-GRP78 signaling by stimulating multiple cancer cell lines with α₂M* in the presence of WT (Leu⁹⁸–Leu¹¹⁵), mutant (K¹¹³–V), or scrambled GRP78 peptides. GRP78 WT peptide suppressed c-MYC induction and decreased α₂M*-dependent phosphorylation of both PDK1 and PLK1. In contrast, GRP78 mutant peptide did not affect α₂M*-mediated c-MYC induction or phosphorylation of PDK1 and PLK1 (Fig. 2D). These results further demonstrate that α₂M* signals specifically through the GRP78 (Leu⁹⁸–Leu¹¹⁵) binding site to induce c-MYC expression.

α₂M* Promotes PLK1 Interaction with c-MYC to Induce Phosphorylation of c-MYC in a PDK1-dependent Manner

To dissect the PDK1/PLK1 signaling pathway, we treated multiple cancer cell lines with the small molecule PDK1 inhibitor BX795 or PLK1 inhibitor BI2536 and then stimulated with α₂M*. Indeed, the PDK1 inhibitor BX795 abolished α₂M*-induced phosphorylation of PDK1 and expression of c-MYC at the protein as well as transcriptional level (Fig. 3, A (first and fifth lanes) and B). Furthermore, like BX795, the PLK1 inhibitor BI2536 eliminated c-MYC protein expression and affected its transcription level but did not affect P-PDK1 (Fig. 3, A (third and fifth lanes) and B). These findings suggest that PLK1 functions as a downstream kinase to PDK1 in α₂M*/CS-GRP78-induced c-MYC expression.

FIGURE 3.

α₂M*/CS-GRP78 signaling is required for the interaction of PLK1 with c-MYC to induce c-MYC phosphorylation. A, immunoblotting (IB) analysis of the indicated cancer cell lines treated with BX795 (2.5 μmol/liter) or BI2536 (10 nmol/liter) for 16 h and then stimulated with α₂M* (100 pm) for 30 min. B, c-MYC transcript analysis in 1-LN prostate cancer cell line treated with BX795 (2.5 μmol/liter) or BI2536 (10 nmol/liter) for 16 h and then stimulated with α₂M* (100 pm) for 30 min. C, immunoprecipitation (IP) analysis of endogenous PDK1 and P-PLK1 in 1-LN prostate cancer cell line with or without BX795 (2.5 μmol/liter) or BI2536 (10 nmol/liter) for 16 h or C38 mAb (50 μg) for 6 h and then stimulated with α₂M* (100 pm) for 30 min. D, immunoprecipitation analysis of endogenous PLK1 and c-MYC in 1-LN prostate cancer cell line with or without BX795 (2.5 μmol/liter) or BI2536 (10 nmol/liter) for 16 h or C38 mAb (50 μg) for 6 h and then stimulated with α₂M* (100 pm) for 30 min. E and F, immunoprecipitation and in vitro kinase assay using recombinant c-MYC and immunoprecipitated endogenous PLK1 from 1-LN Scr-siRNA and si-c-MYC cells in the presence or absence of BX795 (2.5 μmol/liter) or BI2536 (10 nmol/liter) for 16 h or C38 mAb (50 μg) for 6 h and then stimulated with α₂M* (100 pm) for 30 min. Error bars represent S.D.

To determine whether PDK1 is required for the PLK1 activation, we stimulated 1-LN cells with α₂M* followed by PDK1 immunoprecipitation. We demonstrated enhanced phosphorylation of endogenous PLK1 at Thr²¹⁰ that was abolished when cells were treated with BX795 or BI2536 (Fig. 3C). This suggests that PDK1 directly regulates PLK1 in cancer cells. Importantly, cells treated with C38 mAb also showed greatly reduced PLK1 phosphorylation. Together these data indicate that PDK1-induced PLK1 phosphorylation requires α₂M*/CS-GRP78 signaling.

We next investigated whether PLK1 directly regulates c-MYC activation. Through co-immunoprecipitation assays, we showed that α₂M* promoted interaction between PLK1 and c-MYC that is blocked by BX795, BI2536, or C38 mAb (Fig. 3D). We explored whether PLK1 kinase activity is required for the c-MYC phosphorylation and accumulation. We used in vitro kinase assays using endogenous PLK1 pulled down by antibody from α₂M*-stimulated scrambled and c-MYC-silenced 1-LN cells. We observed a robust induction of Ser⁶² phosphorylation of recombinant c-MYC that was blocked in the presence of BX795 or BI2536 (Fig. 3, E and F). Importantly, PLK1 kinase-dependent c-MYC phosphorylation was strongly abolished in cells treated with C38 mAb (Fig. 3, E and F). Phosphorylation of recombinant c-MYC in c-MYC-silenced cells confirms the specificity of the recombinant in vitro kinase assay (Fig. 3F). Thus we not only showed direct phosphorylation of c-MYC by PLK1 but also found that PLK1 activity with respect to c-MYC is crucially dependent on PDK1. Together these data strongly support the hypothesis that α₂M*/CS-GRP78 signaling potentiates PDK1/PLK1/c-MYC signaling in cancer cells.

α₂M*/CS-GRP78 Signaling Axis Phosphorylates Histone H3 Ser¹⁰ in a c-MYC-dependent Manner

To further substantiate the role of c-MYC activation, we stimulated multiple cancer cell lines with α₂M* in the absence or presence of C38 mAb. In α₂M*-stimulated cells, we observed induction of c-MYC Ser⁶² phosphorylation that was accompanied by a similar pattern of phosphorylation of histone H3S10, which is known for the transcriptional activation of the gene. We further showed that C38 mAb and GRP78 WT peptide blocked α₂M*-mediated phosphorylation of c-MYC and P-histone H3S10, whereas c-MYC-silenced cells and GRP78 mutant peptide did not have any further effect (Fig. 4, A, B, and C). Notably, in multiple cancer cell lines, BX795 or BI2536 treatment also inhibited α₂M*-induced phosphorylation of c-MYC and P-histone H3S10 as well as c-MYC accumulation (Fig. 4D). This finding demonstrates that α₂M*/CS-GRP78-mediated c-MYC activity is required for histone H3S10 phosphorylation and suggests a possible role of transcriptional activation of c-MYC target genes.

FIGURE 4.

α₂M*/CS-GRP78 induces histone H3 phosphorylation through PDK1/PLK1/c-MYC signaling. A, immunoblotting analysis of the indicated cancer cell lines treated with C38 mAb (50 μg) for 6 h and then stimulated with α₂M* (100 pm) for 30 min. B, immunoblotting analysis of the indicated proteins in 1-LN Scr-siRNA and si-c-MYC cells stimulated with α₂M* (100 pm) for 30 min. C, various cancer cell lines were stimulated with α₂M* (100 pm) for 30 min in the absence or presence of Scr peptide (100 pm for 1 h), GRP78 WT peptide (100 pm for 1 h), or GRP78 mutant peptide (100 pm for 1 h) and immunoblotted for the respective proteins. D, immunoblotting analysis of the indicated cancer cell lines treated with BX795 (2.5 μmol/liter) or BI2536 (10 nmol/liter) for 16 h and then stimulated with α₂M* (100 pm) for 30 min.

Role of α₂M*/CS-GRP78 Signaling in c-MYC-dependent Gene Regulation

We next sought to determine whether α₂M*-mediated c-MYC induction activates specific sets of target genes that are characteristic of c-MYC transformed cells. To achieve that, we performed human MYC target gene PCR array analysis of α₂M*-stimulated 1-LN cells in the presence or absence of C38 mAb. α₂M* significantly induced expression of c-MYC, FOSL1, and ID2 genes among those genes that were up-regulated (Fig. 5A). Importantly, C38 mAb suppressed up-regulation of these genes by α₂M*, demonstrating a role for CS-GRP78 activation in regulating the c-MYC target genes.

FIGURE 5.

α₂M*/CS-GRP78 signaling is required for the transcriptional activation of c-MYC target genes. A, heat map representation of gene expression -fold changes in a human c-MYC target gene PCR array. 1-LN cells were treated with C38 mAb (50 μg) for 6 h and then stimulated with α₂M* (100 pm) for 30 min. Only genes that were differentially expressed (p ≤ 0.05 and -fold change ≥2) are represented. B, quantitative RT-PCR analysis of FOSL1 and ID2 genes in α₂M*-stimulated scrambled and si-c-MYC 1-LN cells. C, 1-LN cells treated with C38 mAb (50 μg) or control IgG (50 μg) for 6 h and then stimulated with α₂M* (100 pm) for 30 min to quantify the transcript level of the c-MYC target genes FOSL1 and ID2. D, 1-LN prostate cancer cell line treated with BX795 (2.5 μmol/liter) or BI2536 (10 nmol/liter) for 16 h and then stimulated with α₂M* (100 pm) for 30 min prior to analysis of transcript levels of FOSL1 and ID2 genes. Error bars represent S.D.

We next validated c-MYC target genes FOSL1 and ID2 by quantitative PCR analysis. α₂M* induced FOSL1 and ID2 transcript levels in a dose- and time-dependent manner (data not shown). Moreover, silencing of c-MYC strongly inhibited FOSL1 and ID2 expression, and α₂M* had no further effect (Fig. 5B). To confirm that α₂M* induces the c-MYC target genes FOSL1 and ID2 through its receptor CS-GRP78, we treated the 1-LN cells with 50 μg of C38 mAb for 6 h and then stimulated with α₂M*. We observed that targeting CS-GRP78 abrogated the α₂M*-induced FOSL1 and ID2 transcription level (Fig. 5C). These results demonstrate that α₂M*/CS-GRP78 signaling is required for the transcriptional activation of c-MYC target genes.

To show the capability of PDK1/PLK1 signaling in regulation of c-MYC target genes, we treated the 1-LN cell line with BX795 or BI2536 inhibitor. Each compound inhibited induction of FOSL1 and ID2 transcripts by α₂M* (Fig. 5D). Together these findings show that α₂M*/CS-GRP78 signaling modulates PDK1/PLK1 signaling to evoke c-MYC target genes.

α₂M*/CS-GRP78 Signaling Is Required for the Transcriptional Activation of FOSL1 and ID2 Genes

To demonstrate direct binding of c-MYC to the FOSL1 and ID2 regulatory sequences, we performed a ChIP assay using an antibody against c-MYC followed by RT-quantitative PCR analysis in 1-LN cells. As expected, α₂M*-stimulated 1-LN cells showed increased c-MYC association at the FOSL1 enhancer and the ID2 E-box region, whereas C38 mAb, BX795, or BI2536 abolished c-MYC binding and reduced its expression (Fig. 6A). To further confirm the specificity of c-MYC binding to FOSL1 and ID2, we used as a negative primer a downstream region within the first intron of FOSL1 and ID2. These results establish that α₂M*/CS-GRP78 signaling recruits c-MYC to E-box elements of the c-MYC-regulated genes FOSL1 and ID2 through its downstream kinase PDK1/PLK1 signaling, thus contributing to malignant progression.

FIGURE 6.

α₂M*/CS-GRP78 signaling mediates H3S10 phosphorylation and contributes to FOSL1 and ID2 transcription in a c-MYC-dependent manner. A, chromatin samples were obtained from 1-LN prostate cancer cell line stimulated with α₂M* (100 pm) for 30 min in the absence or presence of BX795 (2.5 μmol/liter) or BI2536 (10 nmol/liter) for 16 h or C38 mAb (50 μg) for 6 h. ChIP assays were performed with c-MYC antibody. Immunoprecipitated DNA was analyzed by quantitative PCR with primers to amplify the FOSL1 enhancer and ID2 upstream c-MYC binding sites (−1750/−1460). Real time quantitative PCR measurements of the immunoprecipitated DNA of the corresponding gene regions are expressed in thousandths (%) of the DNA inputs and normalized to the negative (−ve) primers. Values are mean ± S.D. for three replicate samples from one representative experiment (n = 3). Error bars represent S.D. B, phosphorylation of H3S10 at the FOSL1 and ID2 upstream element is dependent on α₂M*/CS-GRP78 signaling. ChIP assays were performed with anti-P-H3S10 antibody in 1LN cells as indicated in A. C, c-MYC silencing of 1LN cells alters H3S10 phosphorylation at the FOSL1 and ID2 enhancer. ChIP assays were performed with anit-P-H3S10 antibody in the sh-c-MYC 1LN cells as indicated in A. UT, untreated.

Next we analyzed whether α₂M*/CS-GRP78 signaling contributes to H3S10 phosphorylation at the FOSL1 and ID2 enhancer region. An analysis of the H3S10 phosphorylation signal in α₂M*-stimulated 1-LN cells showed increased phosphorylation of H3S10 at the FOSL1 and ID2 enhancer region, whereas C38 mAb, BX795, or BI2536 inhibited the H3S10 phosphorylation signal and reduced the expression of FOSL1 and ID2 (Fig. 6B). These results demonstrate that α₂M*/CS-GRP78 regulates the transcriptional activation of FOSL1 and ID2 genes with an increase of H3S10 phosphorylation at the enhancer region. Because in c-MYC-silenced cells the transcription level of FOSL1 and ID2 decreased (Fig. 5B), we analyzed whether c-MYC association with the FOSL1 and ID2 enhancer is required for H3S10 phosphorylation. Quantitative ChIP analysis demonstrated that α₂M* did not induce H3S10 phosphorylation at the FOSL1 and ID2 enhancer in c-MYC-silenced cells (Fig. 6C). These data indicate that H3S10 phosphorylation at the FOSL1 and ID2 enhancer is dependent on c-MYC.

α₂M*/CS-GRP78-induced PDK1/PLK1/c-MYC Signaling Drives Cell Proliferation and Tumorigenesis

To address the functional role of α₂M*/CS-GRP78-mediated PDK1/PLK1/c-MYC signaling in cancer cells, we examined the transforming capacity of α₂M* in multiple cancer cell lines by using the soft agar assay. α₂M* potentiated increased colony numbers in a panel of cancer cell lines, whereas BX795 or BI2536 treatment significantly decreased colony number. This result further demonstrates that PDK1/PLK1 signaling is required for α₂M*-induced transformation. Moreover, targeting CS-GRP78 by C38 mAb abolished α₂M*-induced colony growth (Fig. 7A). Consistent with our previous studies (9), α₂M* significantly increased cell proliferation of multiple cancer cell lines, and this was reduced by BX795 or BI2536 treatment (Fig. 7B). This finding further demonstrates the role of PDK1/PLK1 signaling in cell growth. Indeed, C38 mAb treatment also resulted in more effective growth inhibition. Together these data support the conclusion that α₂M*-induced transformation depends on CS-GRP78-mediated PDK1/PLK1 signaling in cell proliferation.

FIGURE 7.

α₂M* binding to CS-GRP78 promotes cell proliferation through PDK1/PLK1 signaling. A, soft agar growth of various cancer cell lines treated with BX795 (2.5 μmol/liter), BI2536 (10 nmol/liter), or C38 mAb (50 μg) and then stimulated with α₂M* (100 pm). Data in the bar graphs represent mean ± S.D. (n = 3). Error bars represent S.D. B, cell proliferation assay showing the panel of cancer cell lines treated with BX795 (2.5 μmol/liter), BI2536 (10 nmol/liter), or C38 mAb (50 μg) and then stimulated with α₂M* (100 pm). C, apoptosis of the indicated cancer cell lines treated with BX795 (2.5 μmol/liter), BI2536 (10 nmol/liter), or C38 mAb (50 μg) and then stimulated with α₂M* (100 pm) was examined by immunoblotting analysis. UT, untreated.

Next, we evaluated the role of α₂M*/CS-GRP78 in regulating PDK1/PLK1 signaling and cancer cell survival. As expected, BX795, BI2536, or C38 mAb induced massive apoptosis as evidenced by strong detection of poly(ADP-ribose) polymerase cleavage (Fig. 7C). As in our previous studies, targeting CS-GRP78 with C38 mAb induces significant apoptosis (23). Taken together these results further support the role of α₂M*/CS-GRP78 signaling in cell proliferation and malignant progression.

Discussion

This study defines a new role for the α₂M*/CS-GRP78 signaling axis as a c-MYC-dependent modifier of chromatin. On the basis of biochemical and functional evidence, we show that α₂M*/CS-GRP78-dependent PDK1/PLK1 signaling is required for the transcriptional activation of a subset of c-MYC target genes and cell proliferation.

Cell surface expression GRP78 and its ligation by α₂M* are clearly implicated in the development of metastatic prostate cancer (19). Furthermore, CS-GRP78 is differentially expressed in cancer cells and stressed endothelial cells, providing a potential opportunity for highly specific therapeutic intervention (7–13). We and others have shown that cell surface GRP78 through its interaction with α₂M* functions as an upstream regulator of PI3K/Akt signaling (3, 23, 37). Recently, our in vivo model suggested that targeting CS-GRP78 by C38 mAb blocks the Akt pathway to prolong the survival of ovarian cancer-bearing mice (15). By using GRP78 WT (Leu⁹⁸–Leu¹¹⁵) and mutant (K¹¹³–V) peptides, we further show that α₂M* induced PDK1/PLK1/c-MYC signaling through the GRP78 primary amino acid sequence Leu⁹⁸–Leu¹¹⁵, thereby demonstrating that this region is essential for signaling and transcriptional activation of c-MYC. Although α₂M*/CS-GRP78 signaling is associated with PI3K/Akt signaling in oncogenesis, our study uncovered another arm of signaling that routes to PDK1/PLK1/c-MYC signaling to activate c-MYC target genes and promote malignant progression. Consistent with previous findings that α₂M*/CS-GRP78 induces PDK1 activation, we now further show that PDK1 induces PLK1 phosphorylation to maintain the cell growth in cancer cells (22, 25). Importantly, the pathway we identified using a chemical and genetic approach with a GRP78 WT and mutant peptide and C38 mAb treatment shows that α₂M*/CS-GRP78 activates PDK1 to function as an upstream regulator of PLK1 for c-MYC induction. This is in contrast to a previous report suggesting that PDK1 induces c-MYC at a post-translational but not transcriptional level (25, 36). Further studies are needed to determine the mechanism by which α₂M*/CS-GRP78 induces the c-MYC transcript level.

Recent reports highlight that phosphorylation of c-MYC at Ser⁶² enhances c-MYC activity through regulation of protein stability in Ras-expressing cells and prostate and breast cancer cells (25, 30, 38, 39). Consistent with previous reports, we showed by immunoprecipitation experiments after stimulation with α₂M* that PLK1 forms a complex with c-MYC and phosphorylates Ser⁶² (25). Moreover, we have demonstrated that C38 mAb reduces c-MYC protein stability by repressing Ser⁶² phosphorylation and abrogates transcriptional activity of c-MYC. According to the current view, phosphorylation at the NH₂-terminal domain of H3 is required to loosen the interaction between DNA and nucleosome and/or to generate a platform to recruit additional regulatory factors as described in the histone code hypothesis (41). It was shown previously that after treatment with growth factors H3 is phosphorylated rapidly at Ser¹⁰ by MDK1/MSK2 (33, 42–44). Our results confirm these previous findings and emphasize that α₂M*/CS-GRP78 phosphorylates histone H3S10 as a result of c-MYC activity and that this is required for the transcriptional activation of genes mediated by c-MYC. It has been estimated that about 11% of cellular genes contain a functional E-box with which c-MYC can associate on the genome (45). In agreement with these data, ChIP analysis in α₂M*-stimulated 1-LN prostate cancer cells revealed that c-MYC recruitment to chromatin requires CS-GRP78 signaling. As noted, c-MYC is necessary to phosphorylate histone H3S10 to activate transcription of its target genes FOSL1 and ID2. This is in agreement with previous findings that phosphorylation of H3S10 is necessary for transcriptional activation of FOSL1 and ID2 genes (33). C38 mAb suppressed c-MYC target gene expression that is dependent on α₂M* treatment, suggesting that α₂M*/CS-GRP78 participates in transcriptional activation of c-MYC-regulated genes.

C38 mAb strongly inhibited the formation of α₂M*-dependent colonies in soft agar. In addition, PDK1 or PLK1 inhibition resulted in decreased colony number. Although our experiments do not exclude the possibility that other signaling pathways might cooperate with c-MYC, our data strongly suggest that α₂M*/CS-GRP78-dependent PDK1/PLK1/c-MYC signaling induces phosphorylation of histone H3S10 that is necessary to regulate key genes required for c-MYC-dependent cell proliferation. Moreover, the main characteristic of the α₂M*/CS-GRP78-induced proliferation is that it is able to induce tumor initiation and progression. This study therefore provides a molecular mechanism for α₂M*/CS-GRP78-mediated c-MYC-associated gene regulation and c-MYC-dependent proliferation. Because mutations that alter c-MYC expression are among the most common found in human and animal cancers (46), it is conceivable that inhibiting PLK1 association with c-MYC and/or targeting CS-GRP78 by C38 mAb might represent a method for the treatment of c-MYC-driven cancers in human.

An intriguing finding of this study is the identification of α₂M*/CS-GRP78-dependent kinase activation upstream to PDK1/PLK1/c-MYC signaling to regulate cell proliferation (Fig. 8). We provide evidence that α₂M*/CS-GRP78 signaling activates PDK1 to induce PLK1 phosphorylation for the c-MYC activity. We further show here that PLK1 can directly bind to c-MYC and promote transcriptional activation of FOSL1 and ID2 genes by phosphorylating histone H3S10. Regardless of whether or not PDK1/PLK1 signaling regulates c-MYC stability through a similar or distinct mechanism, the regulation of c-MYC by α₂M*/CS-GRP78 signaling immediately suggests a therapeutic approach targeting tumors that are driven by c-MYC. Indeed, our data show a preferential killing by small molecule inhibitors of PDK1 or PLK1, thereby targeting c-MYC in cancer cells. Given that a clinical inhibitor of c-MYC is not available and targeting PLK1 with small molecule inhibitors such as BI2536 had a modest effect in patients with solid tumors (46, 47), this study suggests that therapeutic targeting of CS-GRP78 by C38 mAb may yield a more favorable therapeutic index in c-MYC-associated tumors.

FIGURE 8.

A schematic diagram illustrates that α₂M*/CS-GRP78 signaling drives cell proliferation in a PDK1/PLK1 signaling-dependent manner. α₂M*/CS-GRP78 signaling functions as an upstream regulator of PDK1/PLK1/c-MYC signaling to promote proliferation. Targeting CS-GRP78 with C38 mAb suppresses the α₂M*-mediated signaling and c-MYC target genes, impairing cell proliferation.

In patients with prostate cancer, the levels of both native and α₂M* in serum decrease during disease progression, whereas CS-GRP78 is often found to be overexpressed in human cancer cells when compared with the normal cells (9, 48). Notably, we showed previously that α₂M* increases GRP78 expression in cancer cells (40). Prostate cancer cells should readily bind α₂M* from serum, thus activating CS-GRP78 to promote metastasis with poor survival. This notion is consistent with a recent report from Mandelin et al. (19) showing in vivo that cell surface occurrence of α₂M* and GRP78 is clearly implicated in the development of prostate cancer bone metastasis. In particular, the pathway we identified using multiple approaches in different cancer cell lines validates its relevance in human cancers. Importantly, targeting CS-GRP78 by specific monoclonal antibodies inhibits tumor growth in murine xenograft models of various tumors (12, 15, 37). Collectively, CS-GRP78 has attracted much attention as a potential therapeutic target in cancer. We propose that c-MYC can be an alternative pharmacodynamics marker for the evaluation of C38 mAb under preclinical and clinical development.

Author Contributions

U. G. performed all of the studies contained in this manuscript. M. G-G. gave advice with respect to experimental design and supplied the peptides used in some of the experiments. S. V. P. and U. G. designed and interpreted the studies reported here. U. G. and S. V. P. wrote the paper. All authors have read and approved the final version of this manuscript.