Endoplasmic Reticulum Ribosome Binding Protein 1(RRBP1) Overexpression Is Frequently Found

Endoplasmic reticulum ribosome binding protein 1 (RRBP1) overexpression is frequently found in lung cancer patients and alleviates intracellular stress-induced apoptosis through the enhancement of GRP78

Hong-Yuan Tsai1,2,#, Yi-Fang Yang1,2,#, Alex TH Wu 3, Chi-Jen Yang 4, Yu-Peng Liu5, Yi-Hua Jan2,6 ,Chien-Hsin Lee8, Ya-Wen Hsiao2, Chi-Tai Yeh7, Chia-Ning Shen2, Pei-Jung Lu5, Ming-Shyan Huang5, Michael Hsiao2*

1Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei, Taiwan.

2Genomics Research Center, Academia Sinica

3School of Medical Laboratory Science and Biotechnology, Taipei Medical University Hospital, Taipei, Taiwan

4Department of Internal Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan

5Institute of Clinical Medicine, National Cheng-Kung University, Tainan, Taiwan

6Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan

7Institute of Clinical medicine, Taipei Medical University, Taipei, Taiwan

8Department of Pharmacy, Taipei Tzu Chi General Hospital, Taiwan

#These authors contributed equally to this work

Conflict of interest:

The authors declare no conflict of interest

Running Title:

The role of RRBP1 in lung cancer

Keywords:

RRBP1, GRP78, UPR, apoptosis, lung cancer

Footnotes:

*Correspondence: Dr. Michael Hsiao, D.V.M., Ph.D.

Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2,

Nankang Dist., Taipei 115, Taiwan. Phone: +886-2-27871243; Fax:

+886-2-27899931; E-mail:

1

Abstract

Lung cancer is the leading cause of cancer deaths and is the most occurring malignancy worldwide. Unraveling the molecular mechanisms involved in lung tumorigenesis will greatly improve therapy. During early tumorigenesis, rapid proliferating tumor cells require increase activity of ER for protein synthesis, folding and secretion, thereby are subjected to ER stress. Ribosome binding protein 1 (RRBP1) was originally identified as a ribosome-binding protein located on the rough endoplasmic reticulum and associated with unfolding protein response(UPR). In this report, we investigate the role of the RRBP1 in lung cancer. RRBP1 was highly expressed in lung cancer tissue, as compared to adjacent normal tissue samples as assessed by immunohistochemistry (IHC) in lung cancer tissue array (n=87). Depletion of RRBP1 by short-hairpin RNAs caused ER stress and significantly reduced cell viability, tumorigenicity. This effect is associated with a significant reduction in the expression of GRP78. UPR regulator GRP78, an anti-apoptotic protein that is widely upregulated in cancer, plays a critical role in chemotherapy resistance in some cancers. According to our data, cell with high endogenous RRBP1 expression were more resistant to ER stress. Ectopic expression of RRBP1 alleviated apoptosis that was induced by the ER-stress agent tunicamycin, 2-deoxy-D-glucose and the clinical drug doxorubicin by enhancing GRP78 protein expression. A strong correlation was observed between the expression of RRBP1 and GRP78 in tumor biopsies using the database GSE10072. Our data indicated that RRBP1 may involve in the regulation of mRNA stability of UPR components ATF6 and GRP78. Taken together, RRBP1 could alleviate ER stress and help cancer cell survive. RRBP1 is critical for tumor cell survival, which may make it a useful target in lung cancer treatment and a candidate for the development of new targeted therapeutics.

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Introduction

Lung cancer is the leading cause of cancer deaths worldwide in both men and women. The high incidence of lung cancer is considered to be a consequence of smoking, environmental exposures, genotoxic and family history. Long term expose to carcinogen may induce tumorigenesis. Non small cell lung cancer (NSCLC) accounts for 80 % of all lung cancers worldwide (1). Adenocarcinoma is one of the main types of NSCLC and accounts for 40% of all lung cancers (2). Patients with stage IA disease are treated with surgical resection resulting in 5-year survival of 70% (3), Whereas in stage IV, the 5-year survival is less than 1%. (4). The achievement of early diagnosis and treatment has improved the patients’ outcome. During the early stage of tumor development, cancer cells with high proliferation rates require the increased activity of ER protein folding, assembly, transport and subjected to ER stress. During early tumorigenesis, this adaptive stress response promotes the unfolded protein response (UPR) and initiates the activation of survival cascade mechanisms (5).The activation of the UPR would affect tumorigenesis and help tumor cell survive from ER stress. There is inconclusive evidence of UPR activation mechanisms in human lung tumorigenesis until now. Thus, unraveling the molecular mechanisms involved in lung tumorigenesis will provide novel insight to improve therapy.

The ER protein RRBP1 was originally identified as a ribosome-binding protein on the rough endoplasmic reticulum, which is one the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells (6). RRBP1 has been studied in yeast, where it is a member of the endoplasmic reticulum (ER) stress response and associated unfolded protein response (UPR). In yeast, expression of RRBP1 results in an increase in the levels of KAR2 mRNA, which sequence shares homology with the mammalian protein BiP/GRP78 (7). In a recent report, RRBP1 was recognized as a potential protein marker for colorectal cancer (8). In lung cancer database, RRBP1 is overexpressed in lung cancer compared to adjacent normal tissue. However, there are limited reports of RRBP1 involved in UPR pathway related to cancer development until now.

UPR is involved in signaling pathways emanating from ER. There are three ER stress sensors are IRE-1 (inositol-requiring protein 1), PERK (PKR-like ER kinase), and ATF6 (activating transcription factor). The activation of these sensors is dependent on their dissociation from the ER chaperone BIP/GRP78 (9, 10) GRP78 is a master regulator of the UPR, resides primarily in the ER of mammalian cells and has been implicated in promoting tumor proliferation, metastasis, drug resistance, and apoptosis. (11, 12). GRP78 is frequently overexpressed in lung cancer but not in normal lung tissue (13, 14). GRP78 expression can be induced by treatment with either 2-deoxyglucose (a hypoglycemia-inducing agent) or tunicamycin (an inhibitor of protein glycosylation) (15). These two agents have been shown to induce antitumor effects in vitro and in vivo in combination with chemotherapy (16, 17). Knockdown of GRP78 enhanced the tunicamycin and 2-deoxyglucose- induced increase in apoptosis, as compared with a luciferase shRNA control (18, 19, 20).

In this study, we aimed to investigate the role of the RRBP1 in lung cancer. We detected the expression of RRBP1 proteins in lung cancer and its adjacent normal lung tissue. Depletion of RRBP1 by short-hairpin RNAs caused ER stress and significantly reduced cell viability. Ectopic expression of RRBP1 could alleviated ER stress and protected tumor survive in vitro. We investigated the relationship between the expression of RRBP1 and GRP78 in order to explore the role of RRBP1 in tumorigenesis of lung cancer.

Results

Elevated RRBP1 expression in lung adenocarcinomas cells and patients

To investigate whether RRBP1 plays a role in NSCLC, we first examined the expression of RRBP1 using a tissue array, which contained 87 clinically annotated NSCLC samples. The staining signal of RRBP1 was mainly distributed in the cytoplasm and showed a wide range of expression levels that varied between NSCLC samples (Fig. 1A and B). To elucidate the specificity of RRBP1 antiserum, we used rabbit IgG as isotype control for immunohistochemistry analysis. No signals were detected in the tumor regions using rabbit IgG (Fig S3A). Based on the expression of RRBP1, the NSCLC specimens were classified into four groups: level 0 (negative staining), level 1 (0%- 20% of tumor cells stained), level 2 (20%-50% of tumor cells stained), and level 3 (>50% of tumor cells stained). Statistical analysis showed no significant correlation between RRBP1 expression and tumor stages. However, there was a trend of higher expression of RRBP1 in the early tumor stages (stage I, II and III), which could be observed from the tissue array analysis (Fig. 1C). In addition, the tissue array data demonstrated that RRBP1 was commonly expressed in the tumor regions, but was rare in normal lung tissues (Fig. 1A).. This result was confirmed by performing qPCR analysis on 12 paired normal-tumor clinical specimens, which demonstrated that RRBP1 mRNA was high expression in lung tumors compared with normal tissues (Fig. 1D). Overexpression of RRBP1 in lung tumors was further supported by data analysis from the GEO database (GSE7670) (GSE10072), which showed higher RRBP1 mRNA levels in lung adenocarcinoma than in normal lung tissue (Fig. S1 and S2). In addition, we analyzed RRBP1 levels in 4 N-T paired specimens and found that RRBP1 was overexpressed in 75%(3/4) of lung tumor samples (Fig 1E). The expression profiles of RRBP1 mRNA and protein were examined by qPCR and Western blotting in 8 lung cancer cell lines (Fig. 1F and G). Consistent with the tissue array data, RRBP1 expression was highly variable in lung cancer cell lines. We then treated 5 lung cancer cell lines with 2μΜ tunicamycin and found that high endogenous RRBP1 expression displayed resistance to ER stress agent tunicamycin (Fig. 1H). We concluded that overexpression of RRBP1 might suggest its important role for the survival and maintenance of tumor cells during tumorigenesis

Ectopic RRBP1 expression enhances growth of human lung cancer xenografts

The data described above suggested that RRBP1 may have effects on the maintenance of lung cancer growth and survival in vivo. To test this hypothesis, we generated stable PC13 cell lines that expressed either pCDNA as a control or pCDNA/RRBP1 (Fig. 2A). These cell lines were then injected subcutaneously into NSG mice and allowed to grow as xenografts, and the tumor growth was monitored each week. The tumor size and growth curves of RRBP1-overexpressing tumors were comparable to control tumors (Fig. 2B, 2C). There was approximately a 2-fold increase in tumor size in RRBP1-overexpressed cells. The weight of the xenografts was measured, and the RRBP1-overexpressing tumors showed a significant increase (2-fold) compared to control tumors (Fig. 2D). In addition, in vitro tumorigenicity assays were performed on these two cell lines using a soft agar assay. RRBP1-overexpressing cells showed little more colonies as compared to the control cells (Fig. 2E). These data support the findings above and show that RRBP1 plays an important role during tumor development and helps tumor cells to survive.

Knockdown of RRBP1 expression in high-expressing NSCLC cells significantly reduces cell viability and tumorigenicity in vitro

Base on our tissue array screening and xenograft growth in vivo, our data demonstrated that RRBP1 expression is increased in human lung cancer, as compared to normal alveoli. Therefore, we assessed whether RRBP1 expression may influence the growth characteristics and cell viability of human lung cancer cells. To assess this possibility, we used shRNA lentiviruses to specifically knockdown RRBP1 expression in A549 and CL1-5 cells. A549 and CL1-5 cells were infected with either RRBP1 shRNA containing lentiviruses or luciferase shRNA containing lentiviruses as a control. The RRBP1 knockdown efficiency of each of the individual shRNA clones was analyzed by western blotting (Fig. 2F and G). shRNA clones #2 and #5 inhibited RRBP1 expression at >70% at both the RNA and protein levels, as compared to the control shRNA. Cell growth and cell viability were analyzed by CellTiter-Glo. Cell viability was significantly decreased in the RRBP1-depleted cells, as compared to the cells with the luciferase shRNA (Fig. 2H and I). In addition, RRBP1 silencing significantly decreased tumorigenicity, as measured by a soft agar assay (Fig. 2J). Take together, the loss of RRBP1 reduced cell viability and tumorigenicity in lung cancer cells. Therefore, RRBP1 is essential for tumor survival and maintenance of tumor growth.

RRBP1 depletion significantly reduces tumorigenesis in vivo

To investigate the impact of RRBP1 depletion on tumorigenesis, we subcutaneously injected the cells expressing RRBP1 shRNA or luciferase shRNA into NSG mice. The tumor size and growth curves of RRBP1-depleted tumors were monitored and compared to control (Fig. S4B and C). Knockdown of RRBP1 significantly reduced tumor growth by 75% compared to control tumors (Fig. S4D). For orthtopic lung model, we establish A549-Luc (luciferase) stable clone. A549-Luc-scramble, A549-Luc-shRRBP1 cells were injected into the left lateral thorax of each mouse. Luminescence image was monitored and quantified by noninvasive bioluminesence system. The total luminescence counts are almost equal in RRBP1-depleted and scramble control cells at first day (Fig. 3B and C). At day 49 post-injection, total luminescence counts in scramble control mice were 20-fold higher than in RRBP1-depleted tumor in mice (Fig. 3D and E) and RRBP1-depleted tumor size were significantly reduced as compared to scramble control (Fig. 3F).

Downregulation of RRBP1 activates the p38 and JNK pathway and exacerbates tunicamycin-induced apoptosis by downregulating GRP78 expression in human NSCLC cells

ER stress can activate the MAPK family signaling pathway. Therefore, we investigated that RRBP1 knockdown in human NSCLC could trigger JNK (c-Jun N-terminal kinase) and p38 MAPK (mitogen-activated protein kinase) activation (Fig. 4A). RRBP1 knockdown would cause cellular ER stress. We also found that RRBP1 depletion reduced ATF6 and GRP78 mRNA expression but not IRE1 and PERK (Fig. 4B). Next, we investigated how ATF6 and GRP78 were regulated by RRBP1. We used actinomycin D (transcription inhibitor) to treat RRBP1-depleted cell. The ATF6, GRP78, RRBP1 gene expression level were analyzed by qPCR. GRP78, ATF6 were significantly reduced in RRBP1-depleted cell compared to shluc control (Fig. 4C). We suggested that RRBP1 may involve in ATF6 and GRP78 mRNA stability. However, the molecular mechanism is still unclear.

Furthermore, RRBP1-depleted and luciferase-depleted cells were treated with tunicamycin. The subG1 population was increased in RRBP1-depleted cells, as compared to the controls. Apoptotic cell death was increased from 7.8% to 27.7% and 5.1% to 16.2% in the tunicamycin-treated A549 and CL1-5 cells, respectively (Fig. 4D and E). Western blotting analysis showed that GRP78 protein was significantly decreased in the RRBP1-depleted cells when compared with the controls. Cleaved PARP was also increased in the tunicamycin-treated, RRBP1-depleted cells (Fig. 4F and G). Furthermore, downregulation of GRP78 by GRP78 shRNA enhanced tunicamycin-induced apoptosis, compared to controls (Fig. 4H and I). We suggest that RRBP1-depletion exacerbates tunicamycin-induced apoptosis mediated through GRP78. However, the mechanism is still unclear until now.

RRBP1 and GRP78 expression correlates in human primary lung tumors

Next, we investigated whether RRBP1 and GRP78 expression correlate in NSCLC. We performed immunohistochemistry-based tissue array screening on 87 clinically annotated NSCLCs and found a wide range of GRP78 expression. GRP78 expression is mainly distribution in the cytoplasm of NSCLC. GRP78 overexpression was judged by comparing the staining intensity of NSCLC tumor cells and the adjacent normal alveolar cells (Fig. 5A and B). Overexpression of GRP78 in lung tumors was further supported by data analysis from the GEO database (GSE10072) (Fig. S2B). We examined whether there is an association between RRBP1 and GRP78 expression in human NSCLC tumors. We determined the expression of GRP78 by immunohistochemistry in the same series of patients used for the analysis of RRBP1 expression. Quantification of the immunostaining revealed that RRBP1 expression strongly correlated with GRP78 expression in human lung tumors (Fig. 5C). Using the database GSE10072, statistical analysis showed that RRBP1 mRNA expression was highly correlated with GRP78 mRNA expression in human normal lung and lung tumors (tested by Pearson correlation test, correlation coefficient = 0.42, P<0.05 (Table. S2). The results were consistent with those obtained by immunohistochemistry, showing a significant correlation between the expression levels of RRBP1 and GRP78 (tested by Pearson correlation test, correlation coefficient = 0.281, P<0.05, Fig. 5D).