Y Box-Binding Protein 1 Promotes Epithelial-Mesenchymal Transition, Invasion, and Metastasis of Cervical Cancer via Enhancing the Expressions of Snail.

OBJECTIVE: Y box-binding protein 1 (YB-1) is a potent oncogenic protein. How it regulates Snail in most tumors including cervical cancer is unknown. This article is to study if YB-1 plays a role in cervical cancer via regulating the expression of Snail. METHODS: Immunohistochemical staining of YB-1, Snail, and E-cadherin (E-cad) was performed on tissue specimens including 35 cases of chronic cervicitis (as a control), 35 cases of cervical intraepithelial neoplasm (CIN) I, 35 cases of CIN II/III, 28 cases of unmetastatic cervical squamous cell carcinoma, and 19 cases of metastatic cervical squamous cell carcinoma. RNA interference technique was used to knock down YB-1, E6, and Snail genes. Quantitative polymerase chain reaction, western blot, and transwell experiment were used to detect RNA, protein, and cell invasion of cervical cancer cell lines Hela and C33A, respectively. RESULTS: First, YB-1 knockdown significantly reduced messenger RNA (mRNA) and protein levels of Snail, followed by the increased mRNA and protein levels of E-cad and the decreased invasive ability in both Hela (human papillomavirus [HPV] 18+) and C33A (HPV-) cell lines. Second, YB-1 and Snail protein were correlatively expressed in the group order of metastatic cervical squamous cell carcinoma > unmetastatic cervical squamous cell carcinoma > CINs > cervicitis, with the inverse expression mode of E-cad in the group order, P value less than 0.01, between any 2 groups. Finally, HPV18 E6 knockdown reduced the mRNA and protein levels of YB-1 and Snail in Hela cells. CONCLUSIONS: The results firstly reported that YB-1 whose mRNA expression is regulated by HPV18 E6 promotes epithelial-mesenchymal transition and progression of cervical cancer via enhancing the expressions of Snail, which indicated that YB-1/Snail/epithelial-mesenchymal transition axis could have a potential use in the diagnosis and therapy of cervical cancer metastasis as a cancer marker and molecular target.

Silencing of Y-box binding protein-1 by RNA interference inhibits proliferation, invasion, and metastasis, and enhances sensitivity to cisplatin through NF-κB signaling pathway in human neuroblastoma SH-SY5Y cells.

Y-box binding protein-1 (YB-1), a member of Y-box protein family binding DNA and RNA, has been proposed as a novel marker in multiple malignant tumors and found to be associated with tumor malignancy. Neuroblastoma is an embryonal tumor arising from neuroblast cells of the autonomic nervous system, which is the most common cancer diagnosed in infants. It has been reported that YB-1 is highly expressing in various human tumors including nasopharynx, thyroid, lung, breast, colon, ovary, and prostate cancers. This study aimed to investigate the functional role of YB-1 in neuroblastoma by silencing YB-1 using RNA interference (shRNA) in neuroblastoma SH-SY5Y cells. We found that silencing of YB-1 decreased the proliferation, migration, and invasion of SH-SY5Y cells. At molecular level, inhibition of YB-1 decreased the expression level of PCNA as well as MMP-2 in neuroblastoma SH-SY5Y cells. Also, we discovered that YB-1 silencing sensitized SH-SY5Y cells to cisplatin and promoted the apoptosis induced by cisplatin due to down-regulation of multidrug resistance (MDR) 1 protein via NF-κB signaling pathway. Therefore, we consider that targeting YB-1 is promising for neuroblastoma treatment and for overcoming its cisplatin resistance in the development of new neuroblastoma therapeutic strategies.

MicroRNA-137 chemosensitizes colon cancer cells to the chemotherapeutic drug oxaliplatin (OXA) by targeting YBX1.

The mechanisms underlying oxaliplatin (OXA) resistance in colon cancer cells are not fully understood. MicroRNAs (miRNAs) play important roles in tumorigenesis and drug resistance. However, the relationship between miRNA and OXA resistance in colon cancer cells has not been previously explored. In this study, we utilized microRNA microarray analysis and real-time PCR to verify that miR-93, miR-191, miR-137, miR-181 and miR-491-3p were significantly down-regulated and that miR-96, miR-21, miR-22, miR-15b and miR-92 were up-regulated in both HCT-15/OXA and SW480/OXA cell lines. Blocking miR-137 caused a significant inhibition of OXA-induced cytotoxicity, therefore, miR-137 was chosen for further research. An in vitro cell viability assay showed that knockdown of miR-137 in HCT-15 and SW480 cells caused a marked inhibition of OXA-induced cytotoxicity. Moreover, we found that miR-137 was involved in repression of YBX1 expression through targeting its 3'-untranslated region. Furthermore, down-regulation of miR-137 conferred OXA resistance in parental cells, while over-expression of miR-137 sensitized resistant cells to OXA, which was partly rescued by YBX1 siRNA. The results of this study may aid the development of therapeutic strategies to overcome colon cancer cell resistance to OXA.

FOXO3a Expression Regulated by ERK Signaling is Inversely Correlated With Y-Box Binding Protein-1 Expression in Prostate Cancer.

BACKGROUND: FOXO3a is a member of the forkhead O transcription factors. FOXO3a induces the factors that contribute to cell cycle arrest and is considered a tumor suppressor in several malignant tumors. Y-box binding protein-1 (YB-1) is a multifunctional protein whose high expression is correlated with poor prognoses in various malignant tumors. In the current study, we investigated the relationship between FOXO3a and YB-1 to validate their functional roles in prostate cancer. METHODS: Western blotting and cytotoxicity assays were conducted in prostate cancer cells, LNCaP, and 22Rv1 cells. We also evaluated the protein expressions of FOXO3a and YB-1 in human prostate cancer tissues, using radical prostatectomy specimens. Then, we investigated the correlations between protein expressions and clinicopathologic parameters. RESULTS: We found that both FOXO3a and YB-1 proteins were phosphorylated by ERK signaling, resulting in FOXO3a inactivation and YB-1 activation in LNCaP and 22Rv1 cells. Inversely, inhibition of MEK or treatment with metformin activated FOXO3a through inactivation of ERK signaling and suppressed the viability of LNCaP and 22Rv1 cells in a dose-dependent manner. In immunohistochemical analysis, FOXO3a nuclear expression was inversely correlated with YB-1 nuclear expression (P < 0.0001). Furthermore, high FOXO3a nuclear expression was inversely correlated with a higher Gleason grade (P < 0.0001) and higher preoperative PSA (P = 0.0437). CONCLUSIONS: These results showed that in prostate cancer, FOXO3a, and YB-1 play inverse reciprocal roles as a tumor-suppressor gene and oncogene, respectively, through their master regulator ERK. Prostate 77:145-153, 2017. © 2016 Wiley Periodicals, Inc.

Molecular cloning and characterization of a novel Y-box gene from Sepiella maindroni.

Y-box proteins are a family of highly conserved nucleic acid binding proteins that interact with genome and transcription product to modulate the transcriptional and translational processes. In the present study, a complete mRNA of Y-box binding protein (designated SmYB) was obtained from Sepiella maindroni by amplification of flanking sequences. The full size of SmYB cDNA was 1502 bp, including 99 bp at the 5ꞌ untranslated region (UTR), a 3ꞌ UTR of 821 bp with a poly (A) tail, and an open reading frame of 582 bp, encoding a polypeptide of 193 amino acids with the predicted molecular weight of 16.48 kDa. The conserved cold-shock domain and two known RNA binding motifs identified in SmYB strongly suggested that SmYB was a new member of Y-box proteins. Quantitative real-time PCR was performed to examine the expression of SmYB mRNA in various tissues, embryos, and its temporal expression in liver after cold shock. The mRNA transcript of SmYB was detected in all examined tissues, with the highest expression level in testis and ovary. SmYB was abundant in early developmental stages of S. maindroni embryos but diminished in the late post-embryonic development. In addition, cold-shock treatment upregulated the transcription of SmYB mRNA in liver. These results demonstrated that SmYB is involved in embryonic development of S. maindroni and its tolerance to acute low temperatures.

THE INFLUENCE AND REGULATORY MECHANISM OF Y-BOX BINDING PROTEIN 1 IN OSTEOSARCOMA AND ITS SIGNIFICANCE.

This study quantified the expression of Y-box binding protein 1 (YB-1) by the immunohistochemical method based on pathological paraffin block specimens of aspiration biopsy from patients with osteosarcoma to explore the influence and regulatory mechanism of YB-1 in osteosarcoma and its significance. Patients were divided into two groups with high and low expressed YB-1, and results showed that 7 cases (13.7%) and 18 cases (26.1%) were in level III, and 44 cases (86.3%) and 51 cases (76.9%) were in level IV respectively, and patients with high YB-1 expression quantity had higher malignant tumor degree (p=0.03). Moreover, the tumor necrosis rate induced by chemotherapy in the two groups were 21 cases (41.2%) and 38 cases (51.8%), respectively. By survival analysis, it was found that a 5-year overall survival rate of patients with high YB-1 expression and low YB-1 expression were 61.2% and 76.6%, respectively (p = 0.054), and 5-year event free survival rates were 52.5% and 72.4%, respectively (p = 0.033). Furthermore, metastasis rate of high YB-1 expression and low YB-1 expression were 41.8% and 22.7%, respectively (p = 0.036), indicating that patients with high YB-1 expression had higher pulmonary metastasis rate. Through further study, we discovered that possibly miR-382 plays a regulatory role in YB-1 gene in osteosarcoma.

Overexpression of YB1 and EZH2 are associated with cancer metastasis and poor prognosis in renal cell carcinomas.

Renal cell carcinoma (RCC) is the most common type of kidney cancers in adults, and metastasis represents the major cause of mortality of RCC patients. The Y-box binding protein 1 (YB1) is a multifunctional oncoprotein in various malignancies. Enhancer of zeste homolog 2 (EZH2), a polycomb histone methyltransferase, is a key epigenetic modifier implicated in various cancer metastasis. However, the expression patterns and clinical correlations of both YB1 and EZH2 in RCC remain largely unclear. In this study, the expression of YB1 and EZH2 were examined using immunohistochemistry staining in a study cohort including 165 RCC and 80 tumor adjacent normal tissues. RCC tissues showed a significant higher nuclear expression of YB1 (p < 0.001) and EZH2 (p < 0.001) as compared with the normal counterparts. In addition, YB1 and EZH2 nuclear overexpression were found to be positively associated with RCC stage (p < 0.001 and p = 0.005), Fuhrman tumor grade (p = 0.022 and p = 0.044), and metastasis (p < 0.001 and p = 0.009). Overall survival analysis indicated patients with YB1 (p = 0.004, HR 5.656 (2.006-10.944)) and/or EZH2 (p = 0.006, HR 4.551 (2.124-9.438)) nuclear overexpression correlated with poor survival. More interestingly, YB1 and EZH2 nuclear expression was correlated (p = 0.005). Further studies demonstrated that EZH2 expression was significantly downregulated in YB1 knockdown RCC cell lines. Functionally, YB1 knockdown inhibited RCC invasion in vitro. In conclusion, YB1 and EZH2 expression was correlated and associated with RCC incidence, tumor stage, grade, metastasis, and survival.

YB-1 regulates stress granule formation and tumor progression by translationally activating G3BP1.

Under cell stress, global protein synthesis is inhibited to preserve energy. One mechanism is to sequester and silence mRNAs in ribonucleoprotein complexes known as stress granules (SGs), which contain translationally silent mRNAs, preinitiation factors, and RNA-binding proteins. Y-box binding protein 1 (YB-1) localizes to SGs, but its role in SG biology is unknown. We now report that YB-1 directly binds to and translationally activates the 5' untranslated region (UTR) of G3BP1 mRNAs, thereby controlling the availability of the G3BP1 SG nucleator for SG assembly. YB-1 inactivation in human sarcoma cells dramatically reduces G3BP1 and SG formation in vitro. YB-1 and G3BP1 expression are highly correlated in human sarcomas, and elevated G3BP1 expression correlates with poor survival. Finally, G3BP1 down-regulation in sarcoma xenografts prevents in vivo SG formation and tumor invasion, and completely blocks lung metastasis in mouse models. Together, these findings demonstrate a critical role for YB-1 in SG formation through translational activation of G3BP1, and highlight novel functions for SGs in tumor progression.

The expression level and prognostic value of Y-box binding protein-1 in rectal cancer.

The aims of this study were to simultaneously evaluate the expression of Y-box binding protein-1 (YB-1) in non-neoplastic rectal tissue and rectal cancer tissue, and to collect clinical follow-up data for individual patients. Additionally, we aimed to investigate the developmental functions and prognostic value of YB-1 in rectal cancer. We performed immunohistochemical studies to examine YB-1 expression in tissue samples from 80 patients with rectal cancer, 30 patients with rectal tubular adenoma, and 30 patients with rectitis. The mean YB-1 histological scores for rectal cancer, rectal tubular adenoma, and rectitis tissue specimens were 205.5, 164.3, and 137.7, respectively. Shorter disease-free and overall survival times were found in patients with rectal cancer who had higher YB-1 expression than in those with lower expression (38.2 months vs. 52.4 months, P = 0.013; and 44.4 months vs. 57.3 months, P = 0.008, respectively). Our results indicate that YB-1 expression is higher in rectal cancer tissue than in rectal tubular adenoma and rectitis tissue and that it may be an independent prognostic factor for rectal cancer.

Y-box Binding Protein-1 Is Part of a Complex Molecular Network Linking ΔNp63α to the PI3K/akt Pathway in Cutaneous Squamous Cell Carcinoma.

Cutaneous squamous cell carcinomas (SCCs) typically lack somatic oncogene-activating mutations and most of them contain p53 mutations. However, the presence of p53 mutations in skin premalignant lesions suggests that these represent early events during tumor progression and additional alterations may be required for SCC development. SCC cells frequently express high levels of ΔNp63α and Y-box binding 1 (YB-1 or YBX1) oncoproteins. Here, we show that knockdown of YB-1 in spontaneously immortalized HaCaT and non-metastatic SCC011 cells led to a dramatic decrease of ΔNp63α, cell detachment and death. In highly metastatic SCC022 cells, instead, YB-1 silencing induces PI3K/AKT signaling hyperactivation which counteracts the effect of YB-1 depletion and promotes cell survival. In summary, our results unveil a functional cross-talk between YB-1, ΔNp63α and the PI3K/AKT pathway critically governing survival of squamous carcinoma cells.

Differential expression of the multidrug resistance 1 (MDR1) protein in prostate cancer cells is independent from anticancer drug treatment and Y box binding protein 1 (YB-1) activity.

PURPOSE: The development of a drug-resistant phenotype is the major challenge during treatment of castration-resistant prostate cancer (PC). In solid cancer entities, one of the major contributors to chemoresistance is the multidrug resistance 1 (MDR1) protein. Believed to be involved in the induction of MDR1 expression is the presence of anticancer drugs as well as the Y box binding protein 1 (YB-1). METHODS: Basal as well as drug-induced expression of MDR1 in established PC cell lines was assessed by Western blotting and mass spectrometry. Subsequently, the influence of YB-1 on MDR1 expression was examined via transient overexpression of YB-1. RESULTS: While LNCaP and PC-3 cells showed no detectable amounts of MDR1, the resistance factor was found to be expressed in 22Rv1 cells. Despite this difference, all three cell lines demonstrated similar growth behavior in the presence of the first-line chemotherapeutic agent docetaxel. Incubation of 22Rv1 cells with docetaxel, cabazitaxel, and abiraterone did not significantly alter MDR1 expression levels. Furthermore, overexpression of the MDR1 controlling factor YB-1 showed no impact on MDR1 expression levels. CONCLUSIONS: MDR1 was detectable in the PC cell line 22Rv1. However, this study suggests that MDR1 is of less importance for drug resistance in PC cells than in other types of solid cancer. Furthermore, in contrast to YB-1 properties in other malignancies, MDR1 regulation through YB-1 seems to be unlikely.

Knockdown of Y­box­binding protein­1 inhibits the malignant progression of HT­29 colorectal adenocarcinoma cells by reversing epithelial­mesenchymal transition.

Y­box binding protein­1 (YB­1) has been identified as an oncoprotein in various malignancies. The aim of this study was to investigate the biological role of YB­1 and its association with epithelial­to­mesenchymal transition (EMT) in colorectal cancer (CRC). The expression of YB­1 and three EMT­related proteins (E­cadherin, N­cadherin and vimentin) was analyzed in 80 CRC and matched normal tissue samples, by immunohistochemistry. The results indicated that the expression of YB­1 was higher in CRC tissue samples than that in matched normal controls and was significantly correlated with tumor differentiation, tumor invasion, lymph node metastasis and distant metastases. Furthermore, analysis showed that YB­1 expression was negatively correlated with E­cadherin and positively correlated with N­cadherin and vimentin expression. In vitro assays showed that knockdown of YB­1 inhibited the proliferation, apoptosis resistance, invasion and migration of the HT­29 CRC cell line. Of note, following knockdown of YB­1, E­cadherin expression was elevated whereas N­cadherin and vimentin expression was reduced. Taken together, these results suggest that YB­1 promotes the malignant progression of CRC in part through the induction of EMT, and YB­1 may therefore be a potential novel target for CRC treatment.

Establishment of a novel cell line for the enhanced production of recombinant adeno-associated virus vectors for gene therapy.

Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, pre-existing immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAV-associated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45- and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

Nuclear Y-box-binding protein-1 is a poor prognostic marker and related to epidermal growth factor receptor in uterine cervical cancer.

OBJECTIVE: Y-box binding protein-1 (YB-1) is a member of the cold shock protein family and functions in transcription and translation. Many studies indicate that YB-1 is strongly expressed in tumor cells and is considered a marker of tumor aggressiveness and clinical prognosis. Overexpression of epidermal growth factor receptor (EGFR) has been associated with poor outcomes in cervical cancer. Clinical trials of EGFR family-base therapy are currently being initiated in cervical cancer. Nuclear YB-1 expression correlates with EGFR expression in various types of cancer. However, the clinical significance of nuclear YB-1 expression in different settings, the correlation with EGFR, and the prognostic implications of YB-1 expression in cervical cancer remain elusive. PATIENTS AND METHODS: Nuclear YB-1 expression was immunohistochemically analyzed in tissue specimens obtained from 204 patients with cervical cancer who underwent surgery. Associations of nuclear YB-1 expression with clinicopathological factors such as survival, EGFR expression, and human epidermal growth factor receptor 2 (HER2) expression were investigated. RESULTS: Nuclear YB-1 expression was found in 41 (20.2%) of 204 cases of cervical cancer and correlated with disease stage, tumor diameter, stromal invasion, and lymph-node metastasis. Nuclear YB-1 expression also correlated with EGFR expression (P=0.0114) as well as HER2 expression (P=0.0053). Kaplan-Meier survival analysis showed that nuclear YB-1 expression was significantly associated with poor progression-free survival (P=0.0033) and overall survival (P=0.0003), respectively. CONCLUSION: Nuclear YB-1 expression is a prognostic marker and correlates with EGFR expression in cervical cancer.

Prostaglandin E2 promotes hepatocellular carcinoma cell invasion through upregulation of YB-1 protein expression.

Prostaglandin E2 (PGE2) has been implicated in hepatocellular carcinoma cell invasion. Recently, it was reported that Y box-binding protein 1 (YB-1) is closely correlated with malignancy. This study was designed to examine the mechanisms by which PGE2 increases YB-1 expression and promotes HCC cell invasion. PGE2 greatly enhanced HCC cell invasion through upregulation of the YB-1 protein, and the EP1 receptor is mainly responsible for this regulation. Src and EGFR were both activated by PGE2, which in turn increased the phosphorylation levels of p44/42 MAPK. Src, EGFR and p44/42 MAPK were all involved in PGE2-induced YB-1 expression. Chemical inhibitors and RNAi analysis all confirmed the role of mTOR complex 1 in YB-1 expression induced by PGE2. Furthermore, YB-1 was able to regulate the expression of a series of EMT-associated genes, which indicated that YB-1 could have the potential to control the epithelial-mesenchymal transition process in HCC cells. These findings reveal that PGE2 upregulated YB-1 expression through the EP1/Src/EGFR/p44/42 MAPK/mTOR pathway, which greatly enhanced HCC cell invasion. This study for the first time describes the mechanisms through which PGE2 regulates YB-1 expression and promotes HCC cell invasion.

High expression of Y-box-binding protein 1 is associated with local recurrence and predicts poor outcome in patients with colorectal cancer.

Colorectal cancer (CRC) is one of the most common and fatal malignancies worldwide. Novel prognostic biomarkers are urgently warranted to help improve the treatment of CRC. Y-box-binding protein 1 (YB-1) has been identified as a multifunctional oncoprotein in various malignancies. Our previous study has suggested that YB-1 may promote malignant progression of CRC cells in vitro. However, its clinical and prognostic significance in CRC patients remains unclear. In this study, the expression of YB-1 was examined in 32 fresh CRC tissues using quantitative real-time polymerase chain reaction (qRT-PCR) and in 170 paraffin-embedded CRC tissues using immunohistochemistry. The result of qRT-PCR demonstrated mRNA expression of YB-1 was increased in 26 of 32 (81.25%) of CRC patients. The statistical analysis based on immunohistochemical staining suggested that YB-1 expression was significantly correlated with tumor differentiation, tumor invasion, lymph node metastasis and Dukes' classification (all P<0.05). Furthermore, we found that patients with high YB-1 expression had a poorer prognosis and were more likely to undergo local recurrence, compared to those with low YB-1 expression. We also identified that YB-1 expression, together with lymph node metastasis and Dukes' classification were independent prognostic factors for CRC patients. In conclusion, our study for the first time demonstrated the clinical and prognostic significance of YB-1 in CRC and suggested that YB-1 is of great potential to be an attractive therapeutic target as well as prognostic biomarker for CRC patients.

Peroxiredoxins, thioredoxin, and Y-box-binding protein-1 are involved in the pathogenesis and progression of dialysis-associated renal cell carcinoma.

Patients with end-stage renal disease are exposed to increased oxidative stress and impairment of antioxidant mechanisms. We focused on dialysis renal cell carcinoma (RCC), including epithelial hyperplasia in acquired cystic disease of the kidney (ACDK). We attempted to obtain insight into the carcinogenesis and tumor progression in terms of cellular defense mechanisms associated with oxidative stress by investigating the expression of antioxidant proteins by immunohistochemistry. We evaluated retrospectively 43 cases of dialysis RCC and, as a control group, 49 cases of sporadic RCC. Peroxiredoxin (Prx) 1, 3, 4, 5, and 6 expression in dialysis RCC was positively correlated with the duration of dialysis. In epithelial hyperplasia, in 17 cases of acquired cystic disease of the kidney, Prxs and thioredoxin were highly expressed. Moreover, in dialysis RCC, Prx 3, 4, and 5 immunoreactivity and nuclear expression of Y-box-binding protein-1 were higher than in sporadic RCC. In dialysis RCC, Prx 3, 4, and 5 immunoreactivity positively correlated with the Fuhrman nuclear grade. These data suggest that oxidative stress during dialysis enhances antioxidant activity, with an inhibiting effect on carcinogenesis. Once cancer has developed, antioxidant activity might have a stimulating effect on the progression of dialysis RCC.

Low expression of the X-linked ribosomal protein S4 in human serous epithelial ovarian cancer is associated with a poor prognosis.

BACKGROUND: The X-linked ribosomal protein S4 (RPS4X), which is involved in cellular translation and proliferation, has previously been identified as a partner of the overexpressed multifunctional protein YB-1 in several breast cancer cells. Depletion of RPS4X results in consistent resistance to cisplatin in such cell lines. METHODS: As platinum-based chemotherapy is a standard first line therapy used to treat patients with ovarian cancer, we evaluated the prognostic value of RPS4X and YB-1 at the protein level in specimen from 192 high-grade serous epithelial ovarian cancer patients. RESULTS: Immunohistochemistry studies indicated that high expression of RPS4X was associated with a lower risk of death and later disease progression (HR = 0.713, P = 0.001 and HR = 0.761, P = 0.001, respectively) as compared to low expression of RPS4X. In contrast, YB-1 was not significantly associated with either recurrence or survival time in this cohort. Finally, the depletion of RPS4X with different siRNAs in two different ovarian cancer cell lines reduced their proliferative growth rate but more importantly increased their resistance to cisplatin. CONCLUSION: Altogether, these results suggest that the levels of RPS4X could be a good indicator for resistance to platinum-based therapy and a prognostic marker for ovarian cancer. Our study also showed that RPS4X is an independent prognostic factor in patients with serous epithelial ovarian cancer.

Identification and quantification of newly synthesized proteins translationally regulated by YB-1 using a novel Click-SILAC approach.

Messenger RNA-binding translational regulatory proteins determine in large part the spectrum of transcripts that are translated under specific cellular contexts. Y-box binding protein-1 (YB-1) is a conserved eukaryotic translational regulator that is implicated in cancer progression. To identify specific proteins that are translationally regulated by YB-1, we established a pulse-labelling approach combining Click chemistry and stable isotope labelling by amino acids in cell culture (SILAC). The proteome of TC32 human Ewing sarcoma cells, which robustly express YB-1, was compared with or without YB-1 siRNA knockdown. Cells labelled with light or heavy isotopologs of Arg and Lys were then cotranslationally pulsed with the methionine derivative, azidohomoalanine (AHA). Cells were lysed and newly synthesized proteins were selectively derivatized via a Click (3+2 cycloaddition) reaction to add an alkyne biotin tag. They were then affinity purified and subjected to liquid chromatography-tandem mass spectrometry. This combined Click-SILAC approach enabled us to catalog and quantify newly synthesized proteins regulated by YB-1 after only 45 min of labelling. Bioinformatic analysis revealed that YB-1 regulated proteins are involved in diverse biological pathways. We anticipate that this Click-SILAC strategy will be useful for studying short-term protein synthesis in different cell culture systems and under diverse biological contexts.

Overexpression of Y-box binding protein-1 in cervical cancer and its association with the pathological response rate to chemoradiotherapy.

The aim of this study was to evaluate the expression of Y-box binding protein-1 (YB-1) in nonneoplastic cervical tissue and cervical cancer tissue and to evaluate its relationship with chemoradiosensitivity in the cases of cervical cancer. We performed immunohistochemical studies to examine YB-1 expression among 59 patients with cervical cancer, 30 with cervical intraepithelial neoplasia (CIN), and 30 with cervicitis. The mean YB-1 histological score(HSCORE)values for cervicitis, cervical CIN, and cervical cancer tissues were 22.3, 39, and 84.4, respectively. The mean YB-1 HSCORE value was 80.0 for cervical cancer patients who showed complete pathological response to chemoradiotherapy and 144.3 for cervical cancer patients who showed partial pathological response. Our data showed that the YB-1 expression was the highest in cervical cancer tissue, followed by cervical CIN tissue, and then cervicitis tissues. High YB-1 expression resulted in a lower pathological response rate in patients of cervical cancer than low YB-1 expression did. Our results implied that YB-1 may play a role in the genesis of cervical cancer and that high YB-1 expression decreases the chemoradiosensitivity of cervical cancers.