Functional role of RRS1 in breast cancer cell proliferation

Abstract RRS1 (human regulator of ribosome synthesis 1), an essential nuclear protein involved in ribosome biogenesis, is overexpressed in some human cancers, yet its role in breast cancer remains unclear. Here, we report a functional analysis of RRS1 in breast cancer and its likely mechanism. Immunohistochemistry (IHC) and RT‐qPCR analyses indicated that RRS1 was commonly overexpressed in breast cancer tissues. The copy numbers of RRS1 were higher in tumours compared with those for normal tissues. And there was a significant correlation between copy number and mRNA expression. In addition, RRS1 overexpression was significantly correlated with lymph node metastasis and poor survival. RRS1 mRNA and protein levels were also significantly increased in a panel of human breast cancer cell lines. RRS1 knockdown inhibited proliferation and induced apoptosis and cell cycle arrest in all three cell lines. Furthermore, RRS1 knockdown suppressed the tumour formation and growth of MDA‐MB‐231 cells in nude mice. Additionally, RRS1 knockdown activated p53 and p21 in MCF‐7 cells. A marked increase in the quantity of ribosome‐free RPL11 was detected by Western blot. Moreover, co‐immunoprecipitation (CoIP) experiments showed that RRS1 knockdown activated p53 by facilitating the direct contact of MDM2 and RPL11/RPL5. Taken together, our results suggest that RRS1 may contribute to breast cancer proliferation through RPL11/MDM2‐mediated p53 activation. Therefore, RRS1 may be a promising target for breast cancer therapy.


| INTRODUCTION
Breast cancer is one of the most common malignancies in females worldwide and has an increasing annual incidence; it is commonly considered to be a genetically heterogeneous disease. 1,2 Breast cancer patients usually tend to have different clinical outcome as a result of the genetic alterations in breast cancer; these alterations have been increasingly identified as the critical determinants of breast cancer initiation and progression. 3,4 Growing evidence indicates that targeted therapeutics against gene expression signatures in breast cancer have dramatically improved patient survival in recent decades. 5 Therefore, the elucidation of these factors and their functional roles may help us to understand the progression of breast cancer better and facilitate advances in its treatment.
Jinlian Song and Lin Hou contributed equally to this work. Ribosome biogenesis is the most well-known function of the nucleolus, and it regulates cell growth and cell division. Several genetic diseases, such as cancer and anaemia, are commonly associated with the misregulation of ribosome biogenesis. [6][7][8] Human regulator of ribosome synthesis 1 (RRS1) has been reported in yeast, where it encodes a regulatory nuclear protein consisting of 203 amino acids that is involved in ribosome biogenesis, including maturation, nuclear export and assembly. [9][10][11][12][13] The human RRS1 homologue was subsequently identified from nucleolar extracts. [14][15][16][17] The mammalian RRS1 protein is localized to both the nucleolus and the endoplasmic reticulum and is involved in the endoplasmic reticulum stress response in Huntington disease. 18 Most importantly, RRS1 expression has been recently reported in human cancers, including human colorectal and hepatocellular carcinomas. 19,20 However, the functional role of RRS1 in breast cancer and the mechanistic details of how RRS1 performs this function are currently unknown.
The 5S ribonucleoprotein particle (RNP), as a ribosomal subcomplex, consists of RPL11, RPL5 and 5S rRNA. RRS1 has been shown to regulate the nucleolar localization of the 5S RNP preribosomal complex by directly contacting the complex. 21 And the depletion of RRS1 resulted in an increase in the nucleoplasmic accumulation of both RPL5 and RPL11. It has also been shown that the depletion of RRS1 delays rRNA processing and thus triggers 5S RNP-mediated p53 activation and cellular senescence. 22 Accumulating evidence indicates that all three components of 5SRNP, RPL5, RPL11 and 5srRNA in the nucleoplasm, activate p53 by binding to and inactivating MDM2 in response the nucleolar stress. [22][23][24][25][26] RPL11 is a well-studied participant in the p53 nucleolar stress response pathway. A recent study showed that 5S ribonucleoprotein particle (5S RNP)-mediated p53 activation coupled perturbed ribosomal biogenesis with cell proliferation and cell cycle regulation. 21 Furthermore, the tumour-suppressive role of the 5S RNP-p53 pathway through either MDM2 interaction or Hdm-p53 checkpoint regulation has been widely studied in cancer. 26,27 We thus hypothesize that the 5S RNP-p53 pathway plays an important role in the proliferation of breast cancer upon RRS1 knockdown.
Here, we set out to assess the functional role of RRS1 in breast cancer. We observed that the expression levels of RRS1 were higher in human breast cancer tissues than in paired non-cancerous tissues, and high RRS1 expression levels were associated with lymph node metastasis and poor clinical outcome. Knocking down RRS1 inhibited breast cancer proliferation in vitro and in vivo. Our findings also provide new insights into the RPL11/MDM2/p53 pathway in the proliferation of breast cancer.

| Patient data
All tissue samples, including tumour samples and paired non-cancerous (normal) tissues from the same patients, were collected from 242 female patients with operable primary breast cancer (stages I-III) who underwent breast surgery in 2011 at the Affiliated Hospital of Qingdao University. Clinical information from patients was acquired by reviewing preoperative and perioperative medical records or by written correspondence or telephone. All patients provided informed consent, and all procedures were approved by the ethics board of the Affiliated Hospital of Qingdao University. The ages of the patients at diagnosis ranged from 29 to 70 years, with a median age of 50 years.
The tissues were collected after the diagnosis was confirmed by a senior pathologist. Tumour size, the tumour, node, metastasis (TNM) stage, lymph node status, Ki67 proliferation index, oestrogen receptor (ER) status, progesterone receptor (PR) status and human epidermal growth factor receptor-2 (HER-2) were obtained from reviewing the medical records.

| IHC analysis
All formalin-fixed and paraffin-embedded sections were analysed by IHC. Primary antibodies were used against the following targets: RRS1

| Cell culture and infection
The human breast cancer cell lines MDA-MB-231, BT549 and MCF-7 were cultured in high-glucose DMEM (HyClone, Logan, Aidenbach, Germany) at 37°C. The cells were infected with retroviruses as previously described. 27 RRS1-targeting shRNA (shRNA1 GCTGCCTTCATTGAGTTTA) and a non-targeting shRNA control were expressed via pSuper constitutive expression constructs (Genecard, Shanghai, China).

| CoIP
For the CoIP of endogenous proteins, MCF-7 cells were lysed in cell lysis buffer at 4°C for 30 minutes. 26 The resulting lysate was incubated overnight with an antibody against MDM2 (1:1000; Abcam); protein G sepharose was added, and the sample was agitated for 2 hours at 4°C. Immunoprecipitants were separated by SDS-PAGE after washing with the same buffer and were analysed by immunoblotting with the indicated antibodies.  and protein were extracted. Then, the mice were killed and dissected.

| Statistical evaluation
Each experiment was performed at least three independent times.
Student's t test was used to compare the differences between two groups. Differences in DNA copy numbers between tissue and paired noncancerous normal tissue were tested using the  Figure 1A). In addition, this finding was supported by the observation that RRS1 mRNA levels were also higher in a panel of human breast cancer cell lines than in normal human mammary epithelial cells (HMECs) (P < 0.01, Figure 1B) | 6307 RRS1 protein levels were increased in all three human breast cancer cell lines (P < 0.01, Figure 1C).

| Correlations of RRS1 copy numbers and mRNA levels
To understand the association between the gene copy numbers and mRNA expression, the copy number variations (CNVs) of RRS1 was quantified by real-time PCR in breast tumour tissues and paired tumour-distant normal breast tissues from 24 breast cancer patients.
RRS1 showed significant changes in CNVs in breast tumours, compared with those for normal breast tissues (T/N = 1.705, P = 0.0002) ( Figure 1D). And the relative mRNA expression of RRS1 showed a significant correlation (R 2 = 0.438, P = 0.032) with its relative copy numbers.

| RRS1 expression in breast tissues and its clinicopathological significance in breast cancer patients
Standard immunohistochemical analyses demonstrated that RRS1 was predominantly expressed in the nucleolus of breast tumour epithelial cells (Figure 2A), and 60.7% (147/242) of patients show overexpression of RRS1. When breast cancer patients were stratified by their RRS1 expression, the patients with high RRS1 expression levels exhibited lower DFS than patients with low levels of RRS1 (P < 0.05; Figure 2B). However, a Kaplan-Meier analysis showed that there was no correlation between increased RRS1 levels and OS in patients with breast cancer (P = 0.063). In addition, we analysed the association between the clinicopathological variables and RRS1 expression in 242 breast cancer samples. As shown in Table 1

| RRS1 knockdown suppresses proliferation and induces apoptosis in breast cancer cells
Because RRS1 is highly overexpressed in breast cancer tissues, we

| RRS1 knockdown activated p53 and p21
A previous study revealed that the nucleolus senses various stresses and plays a co-ordinating role in activating p53. 31 The mechanisms underlying how RRS1 knockdown inhibits proliferation in breast cancer are currently unknown; however, because RRS1 is one of the rRNA processing factors in ribosome biogenesis, we speculated that it may affect breast cancer proliferation by activating p53. We knocked down RRS1 in MCF-7 cells and investigated whether RRS1 knockdown activates p53 and its downstream target p21. Our results revealed that RRS1 knockdown increased the levels of p53 and its downstream target p21 and induced cell cycle arrest ( Figure 5).

knockdown-mediated growth inhibition of breast cancer cells
A previous study showed that a common feature of RRS1 knockdown is an increase in the nucleoplasmic accumulation of RPL11. 21 We predicted that ribosome-free RPL11 and MDM2 would be involved in breast cancer proliferation inhibition upon RRS1 knockdown. Thus, we evaluated the levels of ribosome-free RPL11 in MCF-7 cells by isolating ribosomal (Ribo) and non-ribosomal (Non-Ribo) fractions from RRS1 knockdown cells and control cells. Western blot analyses showed that the levels of ribosomal RPL11 decreased significantly in shRRS1 cells, while non-ribosomal RPL11 increased correspondingly ( Figure 6A). In addition, we also measured the levels of MDM2, which interacts with p53 and RPL11/RPL5. The results of co-immunoprecipitation experiments showed that compared with the control, RRS1 knockdown reduced the interaction between MDM2 and p53 ( Figure 6B). In contrast, the interaction between MDM2 and RPL11/RPL5 was enhanced ( Figure 6B). These results indicate that RRS1 knockdown increases the levels of nonribosomal RPL11, which binds to and inhibits MDM2 to activate p53.

| DISCUSSION
Ribosome protein expression is commonly misregulated in cancer. 32,33 In this study, we described for the first time that RRS1 is overexpressed in breast cancer. By analysing breast tissues, we observed that RRS1 mRNA was expressed at higher levels in breast cancer tis- in the nucleoplasm. 38 MDM2, an E3 ubiquitin, inhibits the activity of p53 through proteasome-mediated degradation. 39 The results of this study indicated that RRS1 knockdown caused a marked increase in ribosomefree RPL11 levels and a consequential reduction in ribosomal RPL11 levels in breast cancer cells. In addition, our CoIP experiments also confirmed that MDM2 induced by RRS1 knockdown was in direct contact with RPL11 and p53. In fact, we should further explore the other two components of 5SRNP, RPL5 and 5sRNA, which are critical for p53 activation and cell growth, upon RRS1 knockdown in breast cancer.

| CONCLUSION
In this study, we demonstrate for the first time that the RRS1 and RRS1/RPL11/p53 signal axes are involved in breast cancer proliferation. High RRS1 expression levels were associated with poor breast cancer prognosis, and RRS1 knockdown inhibited breast cancer proliferation in vitro and in vivo. These findings strongly suggest that RRS1 may contribute to breast cancer growth and survival. RRS1 may be characterized as a biomarker and could provide a new possible target for breast cancer treatment.

ACKNOWLEDG EMENTS
We thank Professor Xiaopin Liu for offering cells concerning this study.

CONFLI CT OF INTEREST
The authors declare that they have no competing interests.

Lin
Hou http://orcid.org/0000-0002-6811-2821 F I G U R E 6 RPL11/RPL5 and MDM2 are involved in RRS1mediated growth inhibition. MCF-7 cells were infected with a retrovirus expressing RRS1 (shRRS1) or with a Ctrl vector (shctrl) for 2 days. A, Ribosomal (Ribo) and non-ribosomal (Non-Ribo) fractions were isolated by sucrose gradient centrifugation and immunoblotted using an RPL11/RPL5 antibody. B, Cell lysates were immunoprecipitated with an anti-MDM2 antibody and were immunoblotted with the indicated antibody F I G U R E 5 P53 and p21 are induced by RRS1 knockdown. MCF-7 cells were infected with a retrovirus expressing RRS1 (shRRS1) or with a Ctrl vector (shctrl) for 2 days. Whole-cell lysates were analysed by Western blot. P53 and p21 expression levels were increased by RRS1 knockdown (*P < 0.05 vs shctrl)