Abstract
Activation of c-Myc plays a decisive role in the development of many human cancers. As a transcription factor, c-Myc facilitates cell growth and proliferation by directly transcribing a multitude of targets, including rRNAs and ribosome proteins. However, how to elucidate the deregulation of rRNAs and ribosome proteins driven by c-Myc in cancer remains a significant challenge and thus warrants close investigation. In this report, a crucial role for the HSPC111 (NOP16) multiprotein complex in governing ribosomal biogenesis and tumor growth was determined. It was discovered that enhanced HSPC111 expression paralleled the upregulation of c-Myc and was directly regulated by c-Myc in breast cancer cells. Knockdown of HSPC111 dramatically reduced the occurrence of tumorigenesis in vivo, and largely restrained tumor cell growth in vitro and in vivo. In stark contrast, HSPC111 overexpression significantly promoted tumor cell growth. Biochemically, it was demonstrated that RNA 3′-phosphate cyclase (RTCD1/RTCA) interacted with HSPC111, and RTCD1 was involved in the HSPC111 multiprotein complex in regulating rRNA production and ribosomal biogenesis. Moreover, HSPC111 and RTCD1 synergistically modulated cell growth and cellular size through commanding rRNA synthesis and ribosome assembly coupled to protein production. Finally, overall survival analysis revealed that concomitant upregulation of HSPC111 and RTCD1 correlated with the worst prognosis in a breast cancer cohort.
Implications: Inhibition of HSPC111-dependent ribosomal biosynthesis and protein synthesis is a promising therapeutic strategy to diminish breast cancer tumor progression. Mol Cancer Res; 12(4); 583–94. ©2014 AACR.
Introduction
Breast cancer is by far the most common cancer among women and the leading cause of cancer–related deaths worldwide. The primary tumor and metastases to distant organs often cause significant morbidity and mortality. Numerous studies suggest that the oncogene c-Myc plays a crucial role in the development and progression of breast cancer. Along with its partner protein Max, c-Myc regulates an estimated 10% to 15% of genes in the human genome, and globally reprograms cells and drives proliferation (1–3). Aberrant regulation and overexpression of c-Myc are observed in most tumor types, and the c-Myc signaling is believed to play a critical role in oncogenesis (4–7). A large body of studies have shown that c-Myc is pathologically amplified and/or overexpressed in breast cancers (8, 9), particularly in late-stage tumors (10–12). Its activation seems to be a prognostic marker in predicting recurrence and adverse outcomes in patients with breast cancer (5–7, 12–16).
Enhanced rRNA synthesis and ribosomal biogenesis are commonly seen in human cancers coupled with resultant protein translation and cell-proliferation acceleration (17–19). The effect of c-Myc on driving cell growth is in part due to its role in transcribing rDNA and advancing ribosomal biogenesis (20). The precise mechanisms responsible for c-Myc-controlled ribosomal biogenesis linked to protein synthesis are still largely unknown, such as the cross-talk and coordination among c-Myc–regulated ribosomal proteins, particularly in cancers. A c-Myc target, HSPC111, was recently reported to be overexpressed in breast cancers, and it might have a role in rRNA synthesis and ribosomal assembly (21, 22). Moreover, the upregulation of HSPC111 was associated with poor prognosis in patients with breast cancer (21). However, its biologic role in tumor development and progression has not been recognized, and the molecular basis underlying its involvement in rRNA synthesis and ribosomal biogenesis (such as its partners) has not been characterized.
Here, we embarked on the biologic effects of the HSPC111 multiprotein complex on ribosomal biogenesis and consequential tumor growth. We demonstrated that HSPC111 knockdown largely inhibited cell growth of MDA-MB-231 breast cancer cells in vitro and in vivo. HSPC111 protein was identified to be predominantly localized in nucleus, and was certified to modulate ribosomal biosynthesis. In addition, HSPC111 was testified to interact with RNA 3′-phosphate cyclase (RTCD1), an enzyme catalyzing conversion of a 3′-phosphate group into the 2′,3′-cyclic phosphodiester at the 3′ end of RNA (23, 24). Knocking down HSPC111 or RTCD1, in particular knocking down both, largely hampered overall rRNA synthesis and consequential protein translation in cancer cells. Therefore, targeting HSPC111 or its partners might represent a novel approach for breast cancer therapeutics.
Materials and Methods
Cell culture
The human breast cancer cell lines MDA-MB-231, MCF-7, and T47D were purchased from the Cell Resource Center Affiliated to the Chinese Academy of Medical Sciences. All cells were cultured in RPMI 1640 medium with 10% newborn calf serum and penicillin–streptomycin (100 units/mL) at 37°C with 5% CO2.
Quantitative real-time PCR analysis
Total RNAs were performed from cells using TRizol (Invitrogen), and quantitative real-time polymerase chain reaction (qRT-PCR) analysis was assessed with a kit from Promega according to the manufacturer's instruction. A standard curve was constructed to calculate the relative content of interest mRNAs. glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Primer sequences were listed in Supplementary Table S1.
Viral particle infection
MDA-MB-231 cells were infected with lentiviral transduction particles of HSPC111 short hairpin RNAs (shRNA) and nontarget shRNAs (scrambled control; Sigma). HSPC111 shRNA transduction particles include 5 individual constructs, which target 5 different regions of its mRNA. Stable transfectants were obtained 15 days after selection with puromycin (10 μg/mL; Sigma).
siRNA molecule and plasmid transfection
Prevalidated siRNA molecules were used to target RTCD1 or HSPC111 mRNAs. Following the instructions from the manufacturer, MDA-MB-231 cells were transfected with siRNA molecules and nontarget siRNAs (scrambled control) using siPORTTM NeoFXTM Transfection Agent (Ambion). HSPC111 or c-Myc overexpression construct was transfected into MCF-7 cells using Lipofectamine 2000 (Invitrogen). Experiments were performed 48 hours after transfection.
Protein concentration determination and Coomassie Brilliant Blue staining
Cells (5.0 × 105) were harvested into lysis buffer (Solarbio) after washing with cold PBS. Protein concentration determinations were performed with the Lowry protein assay (Solarbio) according to the instructions provided by the manufacturer. Equal amounts of protein (10 μL/lane) were subjected to 10% SDS-PAGE and processed for Coomassie Brilliant Blue staining as described previously (24).
Western blot analysis
Equal amounts of protein (30–50 μg/lane) were subjected to 10% SDS-PAGE and processed for Western blot analysis as described previously (25). Antibodies (Abs) used were anti-c-Myc (1:2,000; Sigma), anti-RTCD1 (1:1,000; Abcam), anti-HSPC111 (1:200) and anti-GAPDH (1:8,000; the latter 2 Abs from Santa Cruz Biotechnology).
Coimmunoprecipitation and mass spectrometry
Cells were lysed with the mammalian protein extraction reagent (Cwbio. Inc.), and the supernatants were incubated with 2 μg anti-HSPC111, or normal immunoglobulin G (IgG). Thereafter, immunoprecipites were collected with the Gamma-Bind A Sepharose beads (GE Healthcare). Immunoprecipitated proteins were eluted with 4× SDS-PAGE sample buffer by boiling for 5 minutes, and then assessed by Western blot analysis. The proteins within the purified immunoprecipitates were also identified by MALDI-TOF MS (Bruker Daltonics) following the standard procedures (26, 27).
Immunofluorescent staining
Cells were fixed by formaldehyde followed by remobilization with Triton X-100. Thereafter, cells were blocked for 1 hour with 1% Fetal calf serum (FCS) in PBS, and were incubated for 2 hours at room temperature with the anti-HSPC111 rabbit antibody (1:200) and anti-RTCD1 mouse antibody (1:200). Cells were then washed 3 times with PBS followed by incubation with dylight 594-affinipure goat anti-rabbit secondary antibody and dylight 488-affinipure goat anti-mouse IgG secondary antibody (EarthOx, LLC) for 1 hour. After washing with PBS, cells were examined under a confocal laser-scanning microscope. Cell nuclei were counter stained with 4′,6-diamidino-2-phenylindole (DAPI; Molecular Probes).
Cell-proliferation assay
Cell proliferation was determined by cell number counting, the MTT proliferation assay, and the BrdUrd incorporation assay (Roche). Briefly, cells were serum starved overnight and then seeded at a concentration of 5.0 × 104 cells per well in 100 μL culture medium with 1% FCS. Cell culture continued for 48 hours, and then cell growth was assessed following the instructions provided by the manufacturers.
Metabolic labeling of nascent pre-rRNAs with 32P in MDA-MB-231 cells
MDA-MB-231 cells were transfected with HSPC111 or RTCD1 siRNA molecules, or nontarget siRNAs as described above for 48 hours. The culture medium was replaced with phosphate-free medium supplemented with [32P]Orthophosphate (Perkin-Elmer Life Sciences) at a final concentration of 100 μCi/mL, and cells were incubated for 1.5 hours. Thereafter, the culture medium was replaced with nonradioactive medium, and cells were incubated for another 2 hours. Total RNAs were extracted using TRizol from the same number of cells (5.0 × 105) for each group, and finally dissolved in 30 μL ddH2O. An equal volume of total RNAs (20 μL) from each sample was separated with 1% agarose formaldehyde gel, and the radioactive bands were detected by autoradiography.
Animal experiments
All animal care and surgical procedures were approved by the Animal Ethics Committee at RCEES, Chinese Academy of Sciences. Six-week-old immunodeficient (BALB/c nude) female mice were maintained under aseptic sterile conditions. Surgeries were performed under sterile conditions and mice received antibiotics (Gentamycin) in drinking water up to 2 weeks following surgical procedures. MDA-MB-231 cells (4.0 × 106) were orthotopically inoculated into the bilateral mammary fat pads of nude mice. The cells were injected in 1:2 diluted matrigel (BD Biosciences): sterile PBS using a Hamilton syringe. Tumor size was closely monitored with a vernier caliper, and calculated according to the formula π/6 × L × W2 along time course. Mice were sacrificed when tumors reached a size of 1.0 cm3.
Survival analysis
A publicly-available dataset was used to evaluate the role of HSPC111 and RTCD1 in affecting the outcome of patients with breast cancer. This dataset was generated using the Affymetrix oligonucleotide microarray U133a GeneChip, representing 22,000 transcripts, from 286 lymph node–negative patients who had not received adjuvant systemic treatment (28). The normalized mRNA expression data and clinical information were obtained from the NCBI/GenBank GEO database (accession number GSE2034). The 6-year survival rates (for 72 months) were estimated with the Kaplan–Meier method, and the threshold was determined as previously described, that is the expression value 330 for HSPC111 and 1085 for RTCD1 (28).
Quantification of Western blots and statistical analysis
The intensities of autoradiogram were quantified with Image J (NIH, http://rsbweb.nih.gov), and quantified data of each protein were normalized with those of GAPDH. Two-tailed Student t test was used to analyze experimental data between 2 groups. Data were shown in means ± SE. P < 0.05 was considered statistically significant.
Results
A contributive role of HSPC111 in tumor growth
As a crucial transcriptional factor, c-Myc facilitates tumor cell growth through directly transcribing a variety of targets (4–7). A previous study suggested that HSPC111 is a target of c-Myc (25); however, its role in tumorigenesis has not been characterized yet. In this study, to elucidate the role of HSPC111 in breast cancer development and progression, we first assessed the expression of c-Myc and HSPC111 in breast cancer cells. The mRNA level of c-Myc in malignant cell line MDA-MB-231 was considerably increased compared with that in nonmalignant cell lines, T47D and MCF-7 (P < 0.05, Fig. 1A). HSPC111′s promoter has a c-Myc binding site, and its expression is directly transcribed by c-Myc (25). Consistent with the alteration to c-Myc, the mRNA level of HSPC111 was also markedly increased in MDA-MB-231 cells compared with T47D and MCF-7 cells (P < 0.05, Fig. 1A). Similar results were observed for c-Myc and HSPC111 at the protein level evidenced by the Western blot analysis (Fig. 1B), in parallel to the previous findings (25).
Increased c-Myc and HSPC111 expression in malignant breast cancer cells. A, relative expression of c-Myc and HSPC111 in malignant cell line MDA-MB-231 compared with that in nonmalignant cell lines, T47D and MCF-7, assessed by qRT-PCR analysis (n = 5–6). B, Western blot analyses of c-Myc and HSPC111 in MDA-MB-231 cells, MCF-7 cells, and T47D cells. C, Western blot analysis of c-Myc and HSPC111 protein concentrations in MCF-7 cells upon transfection with c-Myc plasmid with or without combination of HSPC111 siRNAs for 48 hours. D, MCF-7 cells were transfected with c-Myc plasmid with or without combination of HSPC111 siRNAs for 48 hours, and then cell growth was assessed with the MTT assay and cell number counting (n = 6).
Increased c-Myc and HSPC111 expression in malignant breast cancer cells. A, relative expression of c-Myc and HSPC111 in malignant cell line MDA-MB-231 compared with that in nonmalignant cell lines, T47D and MCF-7, assessed by qRT-PCR analysis (n = 5–6). B, Western blot analyses of c-Myc and HSPC111 in MDA-MB-231 cells, MCF-7 cells, and T47D cells. C, Western blot analysis of c-Myc and HSPC111 protein concentrations in MCF-7 cells upon transfection with c-Myc plasmid with or without combination of HSPC111 siRNAs for 48 hours. D, MCF-7 cells were transfected with c-Myc plasmid with or without combination of HSPC111 siRNAs for 48 hours, and then cell growth was assessed with the MTT assay and cell number counting (n = 6).
To further delineate the functional link between c-Myc and HSPC111, in other words, to figure out the regulation of c-Myc on HSPC111 and the dependence of c-Myc–driven cell growth on HSPC111 as well, we elevated c-Myc level through forced expression with combination of HSPC111 knockdown in MCF-7 cells. As shown in Fig. 1C, the protein concentration of c-Myc was increased by 34% for cells upon forced expression, and, as a result, HSPC111 was greatly induced by more than 3-fold (lane 3) compared with the control, supporting the regulation of HSPC111 by c-Myc (21, 25). Consequentially, cell growth was significantly promoted by more than 2-fold compared with the control, evidenced by the MTT assay and direct cell number counting (Fig. 1D, P < 0.05). To further confirm the regulation of HSPC111 by c-Myc, we induced exogenous c-Myc expression using a conditionally regulated system by fusing c-Myc and the estrogen receptor hormone-binding domain. Upon induction of tamoxifen (at 1 μmol/L) in MCF-7 cells for 24 hours, c-Myc expression was significantly induced by >3-fold, and, as a result, HSPC111 expression was elevated by >5-fold with consequential great increase (approximately 40%) of cell proliferation (Supplementary Fig. S1 and data not shown). However, for cells transfected with vehicle control construct upon induction of tamoxifen, endogenous c-Myc expression was not significantly affected (data not shown). These results suggested that the upregulation of HSPC111 was because of the induction of exogenous c-Myc rather than endogenous c-Myc for cells transfected with MYC-ER construct. These results together confirmed the regulation of HSPC111 by c-Myc. In support of our finding, Butt and colleagues recently demonstrated that HSPC111 was a target of c-Myc through luciferase reporter assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation assay (21).
When simultaneously overexpressing c-Myc and knocking down HSPC111 (with more than 60% reduction, lane 2 in Fig. 1C), c-Myc–derived cell growth was greatly restrained by approximately 45% compared with the cells with c-Myc overexpression only, as reflected by the MTT assay and cell number counting (Fig. 1D, P < 0.05). It should be noted that cell growth was still significantly simulated in cells with c-Myc overexpression and HSPC111 knockdown, compared with the control (Fig. 1D, P < 0.05). These findings indicated that the ability of c-Myc in driving cell growth partially but not exclusively relied on HSPC111. These data together stressed the functional link between c-Myc and HSPC111 in cancers, and implied that upregulation of HSPC111 driven by c-Myc could be contributive to development and malignancy of breast cancers.
We thus reduced the endogenous expression of HSPC111 in malignant MDA-MB-231 cells through infection of lentivirus-mediated shRNA constructs. We obtained stable transfectants with HSPC111 shRNA expression after selection. As shown in Fig. 2A, The mRNA level of HSPC111 was reduced 63% upon HSPC111-specific shRNAs, and similar results were also observed in the protein level. Consequentially, the cell proliferation of MDA-MB-231 was significantly restrained by 45% characterized with the BrdUrd incorporation assay (P < 0.05, Fig. 2B), and similar results were also reflected by the MTT assay (P < 0.05, Fig. 2B). To decipher the role of HSPC111 in tumorigenesis and tumor progression in vivo, we implanted MDA-MB-231 cells with HSPC111 knockdown into mammary fat pads of nude mice, and tumor formation and growth were closely monitored. We first compared the tumor take rate between the 2 groups. In the scrambled control group, 100% mice (10 of 10) harbored bilateral tumors (for a total of 20 tumors), whereas only 50% mice (5 of 10) developed bilateral tumors and one mouse had a unilateral tumor (for a total of 11 tumors) in the HSPC111-downregulation group. Thus, the tumorigenesis was greatly suppressed by 45% in the HSPC111-downregulation group in comparison to the scrambled control group (55% vs. 100%). As shown in Fig. 2C, the tumor growth for cells with HSPC111 downregulation was substantially repressed along time course compared with the scrambled control from day 24 to day 38 (P < 0.001). The representative tumor image was shown in Fig. 2C, and the final tumor weight was reduced by 86% in mice implanted with HSPC111-shRNA cells compared with the vehicle control (Fig. 2D, P < 0.05).
Tumor growth was modulated by deregulation of HSPC111. A, qRT-PCR and Western blot analyses for the mRNA and protein levels of HSPC111 in MDA-MB-231 cells infected with HSPC111-shRNA viral constructs or scrambled constructs. B, cell proliferation assessed with the BrdUrd incorporation assay and the MTT method (n = 6). C, the growth curves and the representative image of tumors from the 2 groups of mice. D, the final weight of tumors from the mice implanted with HSPC111-knockdown cells or the control mice. E, Western blot analysis of HSPC111 in MCF-7 cells transfected with HSPC111 plasmid for 48 hours. F, MCF-7 cells were transfected with HSPC111 plasmid for 48 hours, and then cell growth was determined by the MTT assay and cell number counting (n = 6).
Tumor growth was modulated by deregulation of HSPC111. A, qRT-PCR and Western blot analyses for the mRNA and protein levels of HSPC111 in MDA-MB-231 cells infected with HSPC111-shRNA viral constructs or scrambled constructs. B, cell proliferation assessed with the BrdUrd incorporation assay and the MTT method (n = 6). C, the growth curves and the representative image of tumors from the 2 groups of mice. D, the final weight of tumors from the mice implanted with HSPC111-knockdown cells or the control mice. E, Western blot analysis of HSPC111 in MCF-7 cells transfected with HSPC111 plasmid for 48 hours. F, MCF-7 cells were transfected with HSPC111 plasmid for 48 hours, and then cell growth was determined by the MTT assay and cell number counting (n = 6).
To confirm the role of HSPC111 in promoting cell proliferation, we thereafter enforced HSPC111 expression in nonmalignant MCF-7 cells. As shown in Fig. 2E, the concentration of HSPC111 protein was induced by >3-fold in cells upon HSPC111 overexpression compared with the vector control. And cell growth was resultantly provoked by approximately 110% evidenced by the MTT assay and by about 70% reflected by cell number counting, compared with the vector control (Fig. 2F, P < 0.05). These results demonstrated that HSPC111 played a vital role in modulating tumor formation and progression, that is upregulation or downregulation of HSPC111 could significantly promote or impede tumor cell growth.
The involvement of RTCD1 in the HSPC111 multiprotein complex
To interpret the mechanism by which HSPC111 regulates tumor growth, we assessed HSPC111′s location and potential interacting protein partners. The Western blot analysis demonstrated that HSPC111 protein was predominantly localized in nucleus, but not in cytoplasm (Fig. 3A), consistent with the previous observation (21). Because of the principal function of nucleus is rRNA transcription and ribosome assembly, these above findings suggested a potential role of HSPC111 in regulating ribosomal biosynthesis and nucleolar integrity. To this end, we thus surveyed the potential binding partners of HSPC111. Immunoprecipitation was thus carried out in the cellular extracts with the HSPC111 antibody, and the immunoprecipitates were purified by TCA-Acetone followed by the mass spectrometry analysis. Based on the mass spectrometry analysis, a few candidate binding partners were determined by selecting the ones precipitated by the HSPC111 Ab after deduction by those precipitated by normal IgG. Among them, RTCD1 was assumed to be the one with great possibility of interacting with HSPC111. RTCD1 was previously identified in the HeLa cell extract, and it is an enzyme that catalyzes conversion of a 3′-phosphate group into the 2′,3′-cyclic phosphodiester at the 3′ end of RNAs (also named RNA 2,3-cyclic phosphate) in an ATP-dependent manner (23, 24). RNA 2,3-cyclic phosphate ends are crucially important in ribosome assembly through regulating RNA-protein binding and RNA stability (27, 29–31). Previous studies have validated that RTCD1-mediated conversion of 2,3-cyclic phosphate ends on rRNAs is indispensable to 18S rRNA biogenesis (32, 33). Because HSPC111 is assumed to play an important role in ribosomal biosynthesis (21), there is likely a physical and functional overlapping between HSPC111 and RTCD1. We therefore assessed the physical interaction between HSPC111 and RTCD1 through Co-IP. We first used the HSPC111 Ab to precipitate its binding proteins in MDA-MB-231 cell extracts. As shown in Fig. 3B (left), RTCD1 was recognized to be coprecipitated with HSPC111. Correspondingly, we used the RTCD1 Ab to precipitate its binding proteins to verify the interaction between RTCD1 and HSPC111. As presented in Fig. 3B (right), HSPC111 was identified to bind with RTCD1. Furthermore, immunohistochemical analysis was used to look into the localization of HSPC111 and RTCD1. As shown in Fig. 3C, HSPC111 and RTCD1 were found to overlap in nucleus for both MDA-MB-231 and MCF-7 cells. These results demonstrated an interaction between HSCP111 and RTCD1, and a potentially important role of RTCD1 in HSPC111 multiprotein complex–elicited biologic functions.
The interaction between HSPC111 and RTCD1. A, Western blot analysis for the distribution of HSPC111 in cytoplasm and nucleus. B, the Co-IP assessment of the interaction between HSPC111 and RTCD1. MDA-MB-231 cell extracts were immunoprecipitated with the anti-RTCD1 Ab or anti-HSPC111 Ab, and coimmunoprecipitated proteins were then determined with anti-HSPC111 Ab or the anti-RTCD1 Ab through Western blot analysis, respectively. The blue arrow indicates RTCD1 (left) or HSPC111 (right) in the IP assessment, and the red arrow indicates the existence of HSPC111 (left) and RTCD1 (right) in the coimmunoprecipitated proteins. C, confocal images indicative of colocalization of HSPC111 and RTCD1 in MDA-MB-231 and MCF-7 cells. Cells were immunostained with antibodies against HSPC111 (red, detected with a dylight 594-affinipure goat anti-rabbit secondary antibody) or RTCD1 (green, detected with a dylight 488-affinipure goat anti-mouse secondary antibody), and were also counter stained with DAPI for nuclei (blue). The original magnification, ×400.
The interaction between HSPC111 and RTCD1. A, Western blot analysis for the distribution of HSPC111 in cytoplasm and nucleus. B, the Co-IP assessment of the interaction between HSPC111 and RTCD1. MDA-MB-231 cell extracts were immunoprecipitated with the anti-RTCD1 Ab or anti-HSPC111 Ab, and coimmunoprecipitated proteins were then determined with anti-HSPC111 Ab or the anti-RTCD1 Ab through Western blot analysis, respectively. The blue arrow indicates RTCD1 (left) or HSPC111 (right) in the IP assessment, and the red arrow indicates the existence of HSPC111 (left) and RTCD1 (right) in the coimmunoprecipitated proteins. C, confocal images indicative of colocalization of HSPC111 and RTCD1 in MDA-MB-231 and MCF-7 cells. Cells were immunostained with antibodies against HSPC111 (red, detected with a dylight 594-affinipure goat anti-rabbit secondary antibody) or RTCD1 (green, detected with a dylight 488-affinipure goat anti-mouse secondary antibody), and were also counter stained with DAPI for nuclei (blue). The original magnification, ×400.
Diminished rRNA synthesis in cells with HSPC111 and RTCD1 reduction
As a master transcriptional factor, c-Myc controls proliferation by directly transcribing a variety of targets, including a number of rRNAs and ribosome proteins (34–36). Upon forced expression of c-Myc (as described in Fig. 1C), the concentrations of 18S and 28S rRNAs were significantly elevated in MCF-7 cells by 3.6- and 4.4-fold, respectively, compared with the vector control cells (Fig. 4A). Meanwhile, HSPC111 downregulation (as described in Fig. 1C) could partially undermine c-Myc–simulated rRNA synthesis (Fig. 4A), in agreement with the results of cell growth (Fig. 1D). These findings supported our hypothesis of a crucial role of HSPC111 (i.e., the HSPC111-RTCD1 complex) in ribosomal biogenesis.
HSPC111 and RTCD1 modulate rRNA synthesis. A, the concentrations of 18S and 28S rRNAs in MCF-7 cells transfected with c-Myc plasmid with or without combination of HSPC111 siRNAs for 48 hours (n = 6). B, the protein levels of HSPC111 and RTCD1 in MDA-MB-231 cells transfected with HSPC111 or RTCD1 siRNAs for 48 hours, determined by Western blot. C, autoradiogram of RNA pulse-labeled with [32P]orthophosphate in HSPC111-low and/or RTCD1-low cells. MDA-MB-231 cells were transfected with HSPC111 and/or RTCD1 siRNAs for 48 hours. The arrows indicate the positions of 28S rRNA and 18S rRNA, respectively. Lane 1, the parental control; lane 2, the scrambled control; lane 3, HSPC111-siRNA; lane 4, RTCD1-siRNA; lane 5, Binal HSPC111 and RTCD1-siRNA. The band intensities for the overall RNAs after normalization to the cell numbers were shown in the bar graph. D, the RNA levels of 18S and 28S rRNAs in MDA-MB-231 cells transfected with HSPC111 and/or RTCD1 siRNAs for 48 hours (n = 6). E, the concentrations of 18S and 28S rRNAs in MCF-7 cells transfected with HSPC111 plasmid for 48 hours (n = 6).
HSPC111 and RTCD1 modulate rRNA synthesis. A, the concentrations of 18S and 28S rRNAs in MCF-7 cells transfected with c-Myc plasmid with or without combination of HSPC111 siRNAs for 48 hours (n = 6). B, the protein levels of HSPC111 and RTCD1 in MDA-MB-231 cells transfected with HSPC111 or RTCD1 siRNAs for 48 hours, determined by Western blot. C, autoradiogram of RNA pulse-labeled with [32P]orthophosphate in HSPC111-low and/or RTCD1-low cells. MDA-MB-231 cells were transfected with HSPC111 and/or RTCD1 siRNAs for 48 hours. The arrows indicate the positions of 28S rRNA and 18S rRNA, respectively. Lane 1, the parental control; lane 2, the scrambled control; lane 3, HSPC111-siRNA; lane 4, RTCD1-siRNA; lane 5, Binal HSPC111 and RTCD1-siRNA. The band intensities for the overall RNAs after normalization to the cell numbers were shown in the bar graph. D, the RNA levels of 18S and 28S rRNAs in MDA-MB-231 cells transfected with HSPC111 and/or RTCD1 siRNAs for 48 hours (n = 6). E, the concentrations of 18S and 28S rRNAs in MCF-7 cells transfected with HSPC111 plasmid for 48 hours (n = 6).
To further test our hypothesis, we looked into the efficacy of overall RNA synthesis affected by the HSPC111–RTCD1 complex. We reduced the endogenous expression of HSPC111 and RTCD1 in MDA-MB-231 cells through transfection of siRNA molecules. As shown in Fig. 4B, the protein level of HSPC111 and RTCD1 was reduced >50% and >60%, respectively. We then surveyed the concentrations of nascent RNAs labeled with 32P from cells. As shown in Fig. 4C, the concentrations of 28S rRNA and 18S rRNA were greatly reduced in cells transfected with HSPC111-specific siRNAs or RTCD1-specific siRNAs compared with those in the parental control and scrambled control cells (P < 0.05). Moreover, a more pronounced decline in the 28S rRNA and 18S rRNA concentrations was observed in cells upon binal HSPC111 and RTCD1 downregulation, compared with the single HSPC111- or RTCD1-knockdown cells (Fig. 4C, P < 0.05). Similar results were demonstrated for the levels of 18S and 28S rRNA validated by the qRT-PCR analysis (Fig. 4D, P < 0.05). In contrast, when we elevated the HSPC111 level in MCF-7 cells, the levels of 18S and 28S rRNAs were markedly increased by 3.8- and 3.4-fold, respectively, compared with the control (Fig. 4E, P < 0.05). These results confirmed a crucial role of HSPC111 in directing rRNA synthesis and assembly, in agreement with the finding from a previous study (21). These results also implied an important role of RTCD1 in modulating rRNA synthesis, consistent with previous studies documenting the involvement of RTCD1 in 18S rRNA biogenesis (37, 38). The further reduction of rRNA concentrations in cells with synergistic HSPC111 and RTCD1 downregulation verified their interactive dependence in commanding rRNA synthesis and assembly.
Attenuated protein synthesis upon HSPC111 and RTCD1 reduction
Elevated rRNA concentrations are commonly seen in human cancers associated with enhanced protein synthesis and accelerated cell proliferation (19, 39). For example, increased rRNA transcription and ribosomal biogenesis were observed in cancers subject to c-Myc activation (17, 18). To this end, we therefore examined the protein concentrations and cell proliferation in MDA-MB-231 cells with HSPC111/RTCD1 knockdown. As shown in Fig. 5A, a significant decline in protein translation as evidenced by the total protein concentrations was observed in HSPC111-low cells or RTCD1-low cells compared with those in the parental control and scrambled control cells (P < 0.05). And the total protein concentrations were further reduced in binal-knockdown (HSPC111-low and RTCD1-low) cells, compared with the individual HSPC111-knockdown or RTCD1-knockdown (P < 0.05). These results were similar to the studies demonstrating diminished protein translation upon c-Myc loss (20). The molecular bases determining cellular shape and size largely rely on the concentrations of cytosolic macromolecules, such as ribosomes and proteins. Because of reduction in rRNA and protein concentrations upon HSPC111 and/or RTCD1 downregulation, the morphology was greatly altered in MDA-MB-231 cells with condensed cellular body (Fig. 5B). The corresponding cellular size was decreased by approximately 22% in HSPC111-low cells and 24% in RTCD1-low cells, compared with the parental control and scrambled control, respectively (P < 0.05), and a further reduction was observed in the double-knockdown cells by 36% (Fig. 5B, P < 0.05,), in agreement with the results of attenuation of RNA synthesis and protein translation (Fig. 4). A similar phenotype was observed to cells upon ribosomal protein S6 (rpS6) deficiency, as the rpS6P−/− β cells had smaller size with diminished insulin production (40).
HSPC111 and RTCD1 modulate protein synthesis, cellular morphology, and cell growth. A, the total cellular protein concentrations of MDA-MB-231 cells transfected with HSPC111 and/or RTCD1 siRNAs. The graph shows quantified data of the total cellular proteins determined by the Lowry protein assay after normalization to the cell numbers (n = 6). B, the images showed the representative morphologic alterations for cells transfected with HSPC111 and/or RTCD1 siRNA molecules for 48 hours. The original magnification, ×100. The cellular size was assessed with a software Image-Pro Plus, and the quantified data for the cellular size were shown in the left panel (n = 20). C and D, cell growth upon HSPC111 and/or RTCD1 downregulation. Cells were transfected with HSPC111 and/or RTCD1 siRNA molecules for 48 hours, and then cell growth was assessed with cell number counting (C) and the MTT method (D; n = 6).
HSPC111 and RTCD1 modulate protein synthesis, cellular morphology, and cell growth. A, the total cellular protein concentrations of MDA-MB-231 cells transfected with HSPC111 and/or RTCD1 siRNAs. The graph shows quantified data of the total cellular proteins determined by the Lowry protein assay after normalization to the cell numbers (n = 6). B, the images showed the representative morphologic alterations for cells transfected with HSPC111 and/or RTCD1 siRNA molecules for 48 hours. The original magnification, ×100. The cellular size was assessed with a software Image-Pro Plus, and the quantified data for the cellular size were shown in the left panel (n = 20). C and D, cell growth upon HSPC111 and/or RTCD1 downregulation. Cells were transfected with HSPC111 and/or RTCD1 siRNA molecules for 48 hours, and then cell growth was assessed with cell number counting (C) and the MTT method (D; n = 6).
Impeded cell growth in response to HSPC111 and RTCD1 reduction
Ribosomal biogenesis and protein synthesis fundamentally determine cell growth (19, 41, 42). Cell growth was remarkably restrained by approximately 25% in MDA-MB-231 cells with HSPC111 knockdown characterized by cell number counting and the MTT assay, compared with the parental control and scrambled control (P < 0.05, Fig. 5C and D). A similar suppression on cell growth was observed in cells upon RTCD1 knockdown with ∼30% decline, reflected by cell number counting and the MTT assay, compared with the parental control and scrambled control (P < 0.05, Fig. 5C and D). Moreover, a further inhibition on cell growth was validated in concomitant-knockdown cells with >35% reduction, compared with the single HSPC111 or RTCD1 knockdown (P < 0.05, Fig. 5C and D). These combined results are in agreement with the notion that c-Myc–induced cell growth is dependent on c-Myc's ability in promoting protein synthesis (20, 43). However, the FACS analysis showed that HSPC111/RTCD1 single or double knockdown did not trigger cell death via either apoptosis or necrosis in MDA-MB-231 cells using FITC-Annexin V and PI staining (data not shown). Thus, these data together highlighted the crucial role of HSPC111 in promoting cell growth through regulating the HSPC111–RTCD1 multiprotein complex–conducted ribosome production and protein synthesis.
High expression of HSPC111 and RTCD1 correlated to poor survival
To recognize the significance of the HSPC111–RTCD1 multiprotein complex in tumor development under the clinical setting, we characterized the association of deregulated HSPC111 and RTCD1 expression with survival in patients with breast cancer. We delineated the Kaplan–Meier survival curves of the relationship between HSPC111 and/or RTCD1 expression and patient survival in a breast cancer cohort database (28). As shown in Fig. 6A, the 6-year (for 72 months) survival curves indicated that upregulated HSPC111 or RTCD1 expression correlated to poor prognosis in patients with breast cancer. More strikingly, concomitant upregulation of HSPC111 and RTCD1 (HSPC111high/RTCD1high) was associated to the worst survival, and concomitant downregulation of HSPC111 and RTCD1 (HSPC111low/RTCD1low) correlated to the best survival, compared with the rest with different expression levels of HSPC111 and RTCD1 (P < 0.001, Fig. 6B). These results suggested that abnormal expression of HSPC111 or RTCD1 was significantly involved in tumor development, and the synergistic upregulation of HSPC111 and RTCD1 exerted more robust impact on tumor progression than one individual gene upregulation only.
High expression of HSPC111 and RTCD1 correlated to poor survival in a breast cancer cohort. Kaplan–Meier survival curves depicting the relationship between HSPC111 and/or RTCD1 mRNA expression and survival rates in a publicly available breast cancer cohort. A, the survival curves related to HSPC111 and RTCD1 mRNA levels, respectively. B, the survival curves describing the synergistic upregulation or downregulation of HSPC111 and RTCD1 mRNA expression.
High expression of HSPC111 and RTCD1 correlated to poor survival in a breast cancer cohort. Kaplan–Meier survival curves depicting the relationship between HSPC111 and/or RTCD1 mRNA expression and survival rates in a publicly available breast cancer cohort. A, the survival curves related to HSPC111 and RTCD1 mRNA levels, respectively. B, the survival curves describing the synergistic upregulation or downregulation of HSPC111 and RTCD1 mRNA expression.
Discussion
Protein translation is concertedly ruled, and deregulation at any step of translational control might predispose cells to transformation or deteriorate tumor progression (20, 42). The oncogene c-Myc enhances protein translation through transactivating diverse targets, such as initiation factors, ribosomal proteins, and rRNAs. Activation of c-Myc is found in a variety of cancers, including breast cancer, and its activation seems to be a surrogate marker for cancers (44–49). The role of c-Myc in driving protein synthesis is in part attributed to promoting ribosome production through transcribing a number of ribosomal proteins (34–36). HSPC111, as a target of c-Myc, was previously characterized as a ribosomal protein residing in a large RNA-dependent nucleolar complex (25). In this study, to the best of our knowledge, we for the first time demonstrated that HSPC111 played a critical role regulating cell growth by involving in rRNA synthesis and ribosomal biogenesis, and the biologic function of HSPC111 essentially relied on its direct binding partner RTCD1.
Ribosomal biogenesis involving the mRNA-to-protein translational machinery is necessary for cell growth and proliferation. Increased ribosome production coupled to elevated protein translation facilitates cell growth and proliferation, and crucially contributes to tumor progression (19, 42). Studies have demonstrated that elevated rRNA synthesis is closely associated with cell transformation and cell proliferation, and a few rRNAs are overexpressed in cancers, such as prostate cancer, gastric cancer, and breast cancer (22, 37). Deregulated control on ribosomal biogenesis linked to enhanced translational capacity significantly contributes to tumorigenesis and tumor aggressivity, as a previous study demonstrated that augmented overall ribosomal production and translational capacity were closely associated with tumor progression in breast cancers (38). In contrast, attenuation on ribosomal biogenesis could repress cell growth and proliferation (38, 50). We here deciphered the function of HSPC111 in governing rRNA synthesis and ribosomal assembly, and also delineated a synergistic interplay between HSPC111 and RTCD1 in rRNA metabolism and ribosomal biogenesis. Cell proliferation and cellular size were remarkably attenuated in cells with HSPC111 or RTCD1 downregulation, likely because of the reduction of overall rRNA synthesis and ribosome production in these cells. A further decline of cell proliferation and cellular size was demonstrated in cells with synergistic HSPC111 and RTCD1 downregulation, supporting a role of their interactive dependence in commanding rRNA synthesis and ribosome assembly.
A previous study suggested that overexpressed HSPC111 was significantly associated with poor prognostic outcome in patients with breast cancer (21). Similar to HSPC111, we here demonstrated that increased expression of RTCD1 was also associated with poor overall survival in patients with breast cancer. Noticeably, concomitant upregulation for both HSPC111 and RTCD1 (HSPC111high/RTCD1high) correlated to the worst prognosis in the breast cancer cohort used in this study. These combined data highlighted the crucial role of HSPC111–RTCD1 in driving cell growth through regulating rRNA synthesis, and also pinpointed a promising direction for cancer therapeutic development by suppressing HSPC111/RTCD1-mediated ribosomal biogenesis.
To summarize, our data suggest that the upregulation of HSPC111 contributes to breast cancer development, and knocking down the endogenous HSPC111 could greatly restrain tumor progression. Importantly, we verified that RTCD1 is a binding partner of HSPC111 in the HSPC111 multiprotein complex formation, and these proteins together synergistically regulate rRNA synthesis and ribosomal assembly, as knocking down HSPC111 or RTCD1, particularly when knocking down both, could profoundly diminish cancer cell growth by repressing ribosomal biogenesis and protein synthesis. Moreover, the synergistic upregulation of HSPC111 and RTCD1 indicated the worst prognosis in a breast cancer cohort. These combined data indicate that interference on the HSPC111–RTCD1 multiprotein complex might exert inhibition on ribosomal biogenesis-driven cell growth. A schematic diagram depicting the possible mechanism responsible for the role of the HSPC111–RTCD1 multiprotein complex in regulating tumor growth is illustrated in Fig. 7. Therefore, our study has implications for the development of therapeutics by targeting HSPC111-mediated ribosomal biosynthesis for the treatment of breast cancers.
A schematic diagram for the role of the HSPC111–RTCD1 multiprotein complex in regulating tumor growth.
A schematic diagram for the role of the HSPC111–RTCD1 multiprotein complex in regulating tumor growth.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: Z. Zhang, Y. Xu, S. Liu
Development of methodology: C. Zhang, C. Yin, L. Wang, J. Ma
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): C. Zhang, L. Wang, S. Liu
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): C. Zhang, C. Yin, L. Wang, S. Zhang, Y. Xu, S. Liu
Writing, review, and/or revision of the manuscript: C. Zhang, L. Wang, Z. Zhang, Y. Xu, S. Liu
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): C. Zhang, Y. Qian
Study supervision: S. Liu
Acknowledgments
We thank lab members for great assistance with experiments and reagents.
Grant Support
This work was supported by a grant from the national “973” program (grant No. 2014CB932000), and grants from the National Natural Science Foundation of China (grant Nos. 21377159 and 81172451).
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