RACK1 promotes lung cancer cell growth via an MCM7/RACK1/Akt signaling complex

MCM7, a member of the miniature chromosome maintenance (MCM) protein family, is crucial for the initiation of DNA replication and proliferation in eukaryotic cells. In this report, we demonstrate that RACK1 regulates cell growth and cell cycle progression in human non-small-cell lung cancer by mediating MCM7 phosphorylation through an MCM7/RACK1/Akt signaling complex. RACK1 functions as a central scaffold that brings Akt into physical proximity with MCM7. Overexpression of RACK1 increases interactions between Akt and MCM7 and promotes Akt-dependent MCM7 phosphorylation, which in turn increases MCM7 binding to chromatin and MCM complex formation. Together, these changes promote DNA replication and cell proliferation. Our findings reveal a novel signaling pathway that regulates growth in non-small cell lung cancer.

Receptor for activated C kinase 1 (RACK1) is a highly-conserved WD40 repeat scaffold protein that belongs to the Trp-Asp (WD) repeat protein family. Individual WD40 repeats can simultaneously interact with multiple signaling molecules, including PKC [26], Src [27][28][29], integrin [30], EphB3 [31], and c-Abl [32], which allows RACK1 to integrate inputs from various signaling pathways [33]. RACK1 therefore plays a pivotal role in many critical cellular processes. Activation of Akt, a Ser/ Thr kinase that participates in many cellular processes by facilitating growth factor-mediated cell survival and blocking apoptosis [34], is associated with tumorigenesis in various human cancers. In addition, a recent study in NSCLC revealed that P-Thr308, but not P-Ser473, which is widely used as a marker of Akt activity, is the major regulator of Akt protein kinase activity [35].
Here, we found that RACK1 was up-regulated in NSCLC, and knockdown of RACK1 inhibited cellular growth and blocked S phase entry. Furthermore, we demonstrated that the oncogenic potential of RACK1 was correlated with MCM7 function. RACK1 regulated the recruitment of MCM7 to chromatin and its interaction with other MCM proteins by regulating its phosphorylation via Research Paper www.impactjournals.com/oncotarget an MCM7/RACK1/Akt signaling complex. These results suggest that RACK1 promotes growth in NSCLC by facilitating interactions between MCM7 and Akt.

RACK1 promotes cellular proliferation by regulating G1/S progression in NSCLC cells
To understand the function of RACK1 in NSCLC cells, we used siRACK1 to knock down its expression in the A549 and H460 NSCLC cell lines. RACK1 knockdown inhibited, while RACK1 overexpression promoted, cell growth and colony formation ( Figure 1A and 1B). Furthermore, flow cytometry revealed that RACK1 knockdown effectively blocked entry into S-phase and reduced the percentage of cells in S-phase, suggesting that RACK1 might regulate the G1 checkpoint ( Figure 1C). To confirm this, we examined the effects of RACK1 on regulators of cell cycle progression at the G1/S boundary. Downregulation of RACK1 decreased cyclinD1 levels, induction of the CDK inhibitor p27, dephosphorylation of Rb, and sequestration of the transcription factor E2F1, but did not alter CDK2, CDK4, or Rb expression, in G1 cells compared to negative controls ( Figure 1D).

RACK1 interacts with MCM7
RACK1 is a scaffold protein that is able to interact with several signaling molecules simultaneously [36]. A two-hybrid yeast assay revealed that RACK1 bound with MCM7, which was a potential downstream regulator of G1/S transition in NSCLC ( Figure 2A). Double immunofluorescence staining in A549 and H460 cells indicated that RACK1 was mainly localized in the cytoplasm but was also expressed to a lesser degree in the nucleus together with MCM7 ( Figure 2B). Both endogenous ( Figure 2C) and exogenous ( Figure 2D) coimmunoprecipitation of RACK1 and MCM7 confirmed their interaction.

RACK1 and MCM7 expression are elevated in clinical NSCLC samples
Next, we performed immunohistochemical staining for the RACK1/MCM7 complex in NSCLC specimens. RACK1 and MCM7 expression were higher in in situ carcinoma and cancer cells than in normal bronchial epithelium cells ( Figure 3A). We then performed immunohistochemical analysis of 150 NSCLC samples using tissue chips and found that RACK1 levels were positively correlated with MCM7 levels (Table 1). Moreover, both RACK1 and MCM7 levels were positively correlated with histological grade, lymphatic metastasis, and tumor TNM stage ( Table 2). A log-rank test showed that NSCLC patients with high RACK1 and MCM7 levels had shorter overall survival ( Figure 3B). To further confirm these findings, we assessed RACK1 and MCM7 mRNA and protein levels in an additional 28 pairs of matched NSCLC and normal lung tissue samples. mRNA and protein levels of both RACK1 and MCM7 were higher in tumor tissue than in the normal lung counterparts ( Figure 3C and 3D). Taken together, these findings indicate that RACK1 and MCM7 are important promoters of NSCLC development and progression.

RACK1 promotes phosphorylation of MCM7
We then examined the mechanism by which RACK1 interacts with MCM7. Interestingly, RACK1 promoted the phosphorylation of MCM7 ( Figure 4A), which increases its activity [37][38][39]. As expected, phosphorylation of MCM7 increased its recruitment to chromatin and MCM complex formation ( Figure 4B and 4C), which are both critical for DNA replication and cellular proliferation [40,41]. These results suggest that RACK1 promotes cellular growth and cell cycle progression via phosphorylation of MCM7.

RACK1 promotes MCM7 phosphorylation indirectly via the Akt signaling pathway
Because RACK1 is a scaffold protein that does not contain catalytic domains, it likely promotes phosphorylation of MCM7 indirectly. Akt also interacted with RACK1 and MCM7; both endogenous ( Figure 5A) and exogenous ( Figure 5B) co-immunoprecipitation experiments confirmed the existence of a ternary MCM7/ RACK1/Akt complex. Furthermore, Akt levels were positively correlated with MCM7 phosphorylation levels ( Figure 5C-5E) and chromatin binding ( Figure 6A-6C). These results suggest that RACK1 indirectly promotes MCM7 activation via the Akt signaling pathway. We therefore investigated whether Akt pathway-induced MCM7 serine/threonine phosphorylation was dependent on RACK1. RACK1 knockdown abolished Akt pathwayinduced MCM7 phosphorylation ( Figure 7A). RACK1 therefore plays an essential role by indirectly promoting MCM7 phosphorylation through the Akt signaling pathway.

DISCUSSION
Here, we demonstrated that RACK1 regulates growth and cell cycle progression in human NSCLC by promoting MCM7 phosphorylation as a component of an MCM7/RACK1/Akt signaling complex. These findings describe a novel mechanism by which promotes proliferation in NSCLC.
Due to its ability to regulate reversible phosphorylation via interactions with protein kinases, phosphatases, and their substrates, RACK1 has been recognized as an important www.impactjournals.com/oncotarget component of signal transduction in several pathways [42]. Overexpression of RACK1 served as a biomarker associated with increased pathological stage, tumor size, and lymph node involvement in pulmonary adenocarcinoma in a recent study [43]. In addition, RACK1 promotes NSCLC by interacting with and activating Smoothened, which in turn mediates Gli1-dependent transcription in NSCLC cells [44]. In this study, we identified a novel signaling pathway by which RACK1 together with MCM7 regulates cell growth and cell cycle progression in NSCLC. MCM7 has long been considered a vital component in the initiation of DNA replication. Numerous studies suggest that phosphorylation of MCM7 is directly correlated with its binding to chromatin and other MCM family members, and decreases in this binding inhibit the helicase activity of MCM7, DNA replication, S-phase entry, and cancer cell growth [38][39][40]. In addition, binding of TPA to PKC activates PKC and its binding to RACK1, which in turn triggers translocation of RACK1 from the nucleus to the cytoplasm, inactivation of MCM7, and disintegration of the DNA replication initiation complex in prostate cancer [41]. However, the specific mechanism by which RACK1 regulates MCM7 activity remains unclear. Here, we found that RACK1 functioned as a central scaffold that brought Akt and MCM7 into close physical proximity in NSCLC cells. Knockdown of RACK1 inhibited the interaction between Akt and MCM7 and decreased Akt-induced phosphorylation of MCM7, resulting in dissociation of MCM7 from chromatin and destruction of the MCM complex. Moreover, RACK1 knockdown inhibited DNA replication and S-phase entry by increasing cell cycle arrest at the G1/S transition checkpoint. In contrast, RACK1 overexpression had the opposite effects. It has also been reported that RACK1 promotes growth in other cancers by affecting key cell cycle regulators. Li et al. reported that downregulation of RACK1 decreased Cyclin D1 expression and induced G1/S cell cycle arrest in pancreatic ductal adenocarcinoma [45]. Lv et al. found that knockdown of RACK1 suppressed Cyclin D1 and CDK6 expression, thereby dramatically increasing G0/G1 phase and reducing S phase cell populations, in glioma [46]. Zhang et al. reported that stable RACK1 knockdown downregulated Cyclin B1 and Cyclin D1 and promoted G1 and G2 phase arrest in oral squamous cell carcinoma [47]. Interestingly, RACK1 has the opposite effect in colon cancer cells; RACK1 overexpression delays passage of HT-29 cells through G1 and mitotic checkpoints by suppressing Src-mediated Sam68 phosphorylation and maintaining the active state of CDK1-cyclinB [48,49].   37 35 In contrast to previous studies, we found here that, although RACK1 was mainly localized in the cytoplasm, it was also expressed to a lesser degree in the nucleus. It has been reported that cAMP/PKA pathway activation may promote the translocation of RACK1 from the cytoplasm to the nucleus [50][51][52][53]. In addition, previous studies have obtained contradictory results regarding the role of RACK1 in regulating Akt activity in different cell types [31,42,[54][55][56]. Our results indicate that RACK1 functions as a protein scaffold that forms a complex by dynamically recruiting MCM7 and Akt. However, there may be other mechanisms by which RACK1 regulates the Akt signaling pathway. In addition, we found that MCM7 phosphorylation was positively correlated with Akt signaling pathway activity in the presence of RACK1, as the MCM7 phosphorylation level was consistent with Akt activity resulting from enhanced IGF-1 stimulation. However, because MCM7 was not confirmed as a direct substrate of Akt, the specific phosphorylation sites and mechanism remain unclear. It is possible that MCM7 phosphorylation is regulated by another protein that also interacts with the Akt signaling pathway. Regardless, immunohistochemical analysis demonstrated that RACK1 expression was positively correlated with MCM7 expression and promoted tumorigenesis in NSCLC.
In conclusion, our results suggest that the scaffold protein RACK1 promotes progression in NSCLC progression by indirectly activating MCM7.

Cell culture and synchronization
Human A549 lung adenocarcinoma cells and H460 large cell lung cancer cells (cell resource center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). For synchronization, cells were starved in medium without serum for 48 h.

Immunoprecipitation and immunoblotting
Cells were harvested in cell lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 2.5 mM sodium pyrophosphate, 1% Na 3 VO 4 , 1 mM PMSF, 0.5 μg/mL leupeptin, 10 μg/mL aprotinin, 1 mM phosphatase inhibitors). Cell lysates were incubated on ice for 30 min and then centrifuged at 12000 rpm for 20 min. The resulting supernatants were collected and assayed in immunoblotting experiments. For immunoprecipitation experiments, the relevant antibodies were added (1 μg/1 mg lysates) and incubated at 4°C overnight. Fifty μL of protein A/G beads were then added and incubated for an additional 4 h at 4°C. The beads were collected, washed 5 times with lysis buffer, and (E) Serum-starved A549 cells were treated with 2.5 or 5.0 µM perifosine as indicated for 1 h prior to the addition of 100 ng/mL IGF-1 for 10 min. MCM7 immunoprecipitates were probed for P-Ser, P-Thr, and MCM7. resuspended in 1 × SDS sample buffer. The samples were then separated by SDS-PAGE and immunoblotted using the relevant antibodies.

Chromatin association assay
The chromatin association assay was performed as previously described [37]. Cells were lysed in 1 mL of Buffer A (15 mM NaC 2 H 3 O 2 , 110 mM KC 2 H 3 O 2 , 2 mM MgC 2 H 3 O 2 , 0.5 mM EGTA, and 20 mM HEPES, pH 7.3). DTT and digitonin were then added to the cell suspension at final concentrations of 2 mmol/L and 50 mg/mL, respectively, and suspensions were placed on ice for 10 min. The suspension was then centrifuged at 1500 g for 10 min at 4°C. Pelleted nuclei were resuspended in hypotonic buffer (Buffer B: 1 mM HEPES, pH 7.5, 0.5 mM EDTA supplemented with 0.5% NP40). The nuclear suspensions were then placed on ice for 15 minutes and laid on top of a 10-mL sucrose cushion (100 mM sucrose and 0.5 mM Tris-HCl, pH 8.5) and centrifuged at 3500 g for 10 minutes at 4°C. The chromatin pellets were suspended in 0.25 mM EDTA (pH 8.0) and sonicated for 10 seconds two times per  sample to produce a chromatin-rich supernatant fraction. Proteins in these fractions were quantitated and analyzed using SDS-PAGE.

Colony formation assay
The A549 and H460 cells were plated in 40-mm dishes 24 h after transfection (1000 per dish) and incubated for 12 days. Medium containing 10% fetal bovine serum was replaced every 4 days. On day 12, the plates were washed with PBS, stained with hematoxylin, and the number of colonies with more than 50 cells was counted.

3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay
Cells were plated in 96-well plates at a concentration of 3000-5000 cells per well 24 h after transfection. To measure cell growth, 20 μL of 5 mg/mL MTT was added to the media and samples were incubated for 4 h at 37°C. The media were then removed, 200 μL of DMSO was added to dissolve the generated deposits, and the absorbance at 490 nm was measured using an automatic microplate reader. The measurement process was performed every 24 h for 5 days to generate a cell growth curve.

Immunofluorescence
Twenty-four hours after they were plated on covered slides, cells were washed 3 times with ice-cold PBS and fixed in 4% paraformaldehyde for 20 min at room temperature. After two PBS washes, the cells were blocked with 3% BSA with 0.2% Triton X-100. Cells were then stained with primary antibodies diluted in 3% BSA overnight at 4°C. After 3 washes in PBS, cells were incubated for 2 h with the secondary donkey anti-mouse (fluorescein isothiocyanate conjugated, 1:300) or goat anti-rabbit (tetramethylrhodamine-isothiocyanatee conjugated, 1:300) antibodies, as appropriate. Nuclei were counterstained with propidium iodide (PI, 50 μg/mL, Sigma).

Flow cytometry
For DNA content analysis, cells were fixed in 70% ethanol overnight at 4°C and then washed with PBS containing 1% BSA. Cells were incubated in 50 μg/mL propidium iodide and 100 μg/mL RNase A for 30 min, and 10000 cells per sample were analyzed using a FACSCalibur cytometer (BD Biosciences).

Yeast two-hybrid screening
The fusion protein pGBKT7-MCM7 contained 719 amino acids from MCM7 and 219 amino acids from bait domain [37]. The construct was transformed into One ShotTM competent cells (Invitrogen, Carlsbad, CA). pAD-RACK1 was constructed in pACT2 in 0.5 mL of polyethylene glycol/LiAc and incubated at 30ºC for 30 minutes. After this initial incubation with plasmid DNA, the cell solution was combined with 20 mL of DMSO and incubated for 15 minutes at 42ºC. The cells were pelleted, resuspended in 1 mL YPD medium, and shaken at 30ºC for 40 minutes. The transformed cells were then pelleted, resuspended in 0.5 mL 0.9% NaCl, and plated onto the appropriate SD agar plate. The transformants were first plated on low stringency SD-Leu/-Trp and medium stringency SD-Leu/-Trp/-His plates. The colonies that grew on those plates were then subjected to the β-galactosidase assay as previously described for 24 and then allowed to grow further on the high stringency SD-Ade/-His/-Leu/-Trp plate.

Patients and specimens
This research was approved by the Human Research Ethics Committee of China Medical University, which is accredited by the National Council on Ethics in Human Research. All patients signed informed consents and were closely monitored during follow-up observations. None of the patients received chemotherapy or radiotherapy prior to operations.

Primary tumor specimens
Primary NSCLC and matched normal lung tissue specimens were obtained from 28 patients who underwent surgical resection at the First Affiliated Hospital of China Medical University between 2010 and 2011. Samples were cut from the resected lungs immediately after removal, frozen in liquid nitrogen, and then stored at −80°C.

Statistics
Immunohistochemistry results were analyzed using the chi-square test and Spearman rank correlation. Kaplan-Meier survival analyses were carried out and compared using the log-rank test. Differences between groups were compared using two-tailed Student's t-test; p values < 0.05 (*) or < 0.01 (**) were considered statistically significant.