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Background: Cyclic-dependent kinase (CDK) 4/6 kinases, as the critical drivers of the cell cycle, are involved in the tumor progression of various malignancies. Pharmacologic inhibitors of CDK4/6 have shown significant clinical prospects in treating hormone receptor-positive and human epidermal growth factor receptor-negative (HR + /HER2-) breast cancer (BC) patients. However, acquired resistance to CDK4/6 inhibitors (CDK4/6i), as a common issue, has developed rapidly. It is of great significance that the identification of novel therapeutic targets facilitates overcoming the CDK4/6i resistance. PARP1, an amplified gene for CDK4/6i-resistant patients, was found to be significantly upregulated during the construction of CDK4/6i-resistant strains. Whether PARP1 drives CDK4/6i resistance in breast cancer is worth further study. Method: PARP1 and p-YB-1 protein levels in breast cancer cells and tissues were quantified using Western blot (WB) analysis, immunohistochemical staining (IHC) and immunofluorescence (IF) assays. Bioinformatics analyses of Gene Expression Profiling Interactive Analysis (GEPIA), Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Cell Line Encyclopedia (CCLE) datasets were applied to explore the relationship between YB-1/PARP1 protein levels and CDK4/6i IC50. Cell Counting Kit-8 (CCK-8) and crystal violet staining assays were performed to evaluate cell proliferation rates and drug killing effects. Flow cytometry assays were conducted to assess apoptosis rates and the G1/S ratio in the cell cycle. An EdU proliferation assay was used to detect the DNA replication ratio after treatment with PARP1 and YB-1 inhibitors. A ChIP assay was performed to assess the interaction of the transcription factor YB-1 and associated DNA regions. A double fluorescein reporter gene assay was designed to assess the influence of WT/S102A/S102E YB-1 on the promoter region of PARP1. Subcutaneous implantation models were applied for in vivo tumor growth evaluations. Results: Here, we reported that PARP1 was amplified in breast cancer cells and CDK4/6i-resistant patients, and knockdown or inhibition of PARP1 reversed drug resistance in cell experiments and animal models. In addition, upregulation of transcription factor YB-1 also occurred in CDK4/6i-resistant breast cancer, and YB-1 inhibition can regulate PARP1 expression. p-YB-1 and PARP1 were upregulated when treated with CDK4/6i based on the WB and IF results, and elevated PARP1 and p-YB-1 were almost simultaneously observed during the construction of MCF7AR-resistant strains. Inhibition of YB-1 or PAPR1 can cause decreased DNA replication, G1/S cycle arrest, and increased apoptosis. We initially confirmed that YB-1 can bind to the promoter region of PARP1 through a ChIP assay. Furthermore, we found that YB-1 phosphorylated at S102 was crucial for PARP1 transcription according to the double fluorescein reporter gene assay. The combination therapy of YB-1 inhibitors and CDK4/6i exerted a synergistic antitumor effect in vitro and in vivo. The clinical data suggested that HR + /HER2- patients with low expression of p-YB-1/PARP1 may be sensitive to CDK4/6i in breast cancer. Conclusion: These findings indicated that a "YB-1/PARP1" loop conferred resistance to CDK4/6 inhibitors. Furthermore, interrupting the loop can enhance tumor killing in the xenograft tumor model, which provides a promising strategy against drug resistance in breast cancer. [ABSTRACT FROM AUTHOR]

In the article, the author examines the properties of Y-box-binding protein (YB-1) and expression of the YBX-1 gene in various malignant tumors and provides the data from her own prospective study in breast cancer patients. YB-1 is a member of the highly conserved family of cold shock proteins with multiple functions in the cytoplasm and cell nucleus. YB-1 is involved in embryogenesis; it ensures cell proliferation and protects cell from the action of various aggressive environmental factors. In adult organisms, YB-1 is involved in a variety of cellular functions that regulate malignant phenotype in several types of tumors. YB-1 is a molecular marker of tumor progression that can be used in clinical practice as both prognostic factor and a target for anticancer therapy. Our prospective clinical study showed that expression of YB-1 mRNA is an independent prognostic factor, as breast cancer patients expressing YB-1 have a lower disease-free survival rate, regardless of the tumor stage and biological subtype. We recommend determining the level of YB-1 mRNA expression as a prognostic test in breast cancer patients. [ABSTRACT FROM AUTHOR]

Purpose: The cold-shock protein Y-box binding protein-1 (YB-1) is associated with more frequent relapse and higher aggressiveness in breast cancer and, notably, is a client protein of estrogen receptor a (ERa). Thus, the authors hypothesized that endocrine therapy using the antiestrogens 4-hydroxytamoxifen (4-OHT) and fulvestrant (FUL) may affect YB-1 expression. Materials and Methods: YB-1 localization in the breast cancer cell line MCF-7 was determined by fluorescence microscopy and GST-ERa pull-down assay. Modulation of YB-1 expression in the presence of 4-OHT and FUL was determined by quantitative RT-PCR as well as by Western blot analysis. Results: YB-1 is primary localized in the perinuclear cytoplasm of MCF-7 cells. YB-1 binds directly to recombinant GST-ERa fusion protein as shown by pull-down assay. Incubation experiments with 4-OHT and FUL demonstrated a strong time- and dose-dependent suppression of YB-1 expression by these antiestrogens. Inhibitory effects were assessed on the level of YB-1 mRNA as well as on the level of YB-1 protein. Conclusion: The data presented here suggest that 4-OHT and FUL therapy targets both proliferative ERa as well as pro-oncogenic YB-1. Thus, 4-OHT's and FUL's anticancer efficacy may play a more global role in breast cancer progression control than originally thought. [ABSTRACT FROM AUTHOR]

Purpose of investigation: The pleiotropic factor Y-box binding protein-1 (YB-1) is clinically correlated with breast cancer patients' poor outcome. Due to the intereference of YB-1 with the estrogen receptor, the authors examined the antiproliferative efficacy of 4-hydroxytamoxifen (4-OHT) and fulvestrant (FUL) in the presence of low and high levels of YB-1. Materials and Methods: The human breast cancer cell line MCF-7 was transfected to overexpress YB-1 protein. 4-OHT and FUL incubation experiments were performed and cell growth behaviour of YB-1 overexpressing MCF-7 cells were compared to unmodified MCF-7 cells expressing endogenous YB-1 levels. Results: The incubation of MCF-7 cells with 100 nM 4-OHT and 1 nM FUL over a period of 120 hours clearly demonstrated the antiproliferative properties of both antiestrogens as expected. Notably, high levels of YB-1 led to an increase of antiproliferative properties in the presence of 4-OHT and FUL. Conclusion: The data presented here demonstrated YB-1 enhances the antiestrogenic efficacy of 4-OHT and FUL. Thus, the group of high level YB-1 mamma carcinoma patients might increasingly benefit from endocrine therapy. [ABSTRACT FROM AUTHOR]

Programmed cell death 1 (PD-1) suppresses T effector functions by inhibiting signaling downstream of the T cell receptor and helping tumor cells escape the immune response. However, the effect and mechanism of cell-intrinsic PD-1 in cancer cells are still unknown. Here, we found that PD-1 is aberrantly upregulated in TNBC patients and cell lines. Cell-intrinsic PD-1 in TNBC cells significantly facilitated tumor growth and metastasis in vitro and in vivo. Further studies indicated that PD-1 effect on TNBC cell growth depends on the cell-intrinsic-PD-1/PD-L1 pathway independent of adaptive immunity. In addition, we further found that the activation of cell-intrinsic PD-1/PD-L1 pathway in TNBC cells is regulated by the gene expression regulator YB-1. Mechanistically, the results of protein degradation analysis, mRNA translationally active analysis, CLICK chemistry and L -azidohomoalanine (AHA) incorporation assays, immunoprecipitation assay and Dual-Luciferase reporter assay showed that YB-1 promotes PD-1 expression through the translational activation pathway. We provide in vitro and in vivo evidence that silencing YB-1 expression in TNBC cells inhibits cell proliferation, tumorigenesis, and metastasis. However, this inhibition can be rescued by simultaneous exogenous expression of PD-1 and PD-L1 proteins. In conclusion, our results identify TNBC cell-intrinsic functions of the PD-1/PD-L1 axis in tumor growth and metastasis; and revealed PD-1/PD-L1 is a critical effector of YB-1-mediated TNBC proliferation and metastasis in vitro and in vivo. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Sox2, a transcription factor and an embryonic stem cell marker, has been implicated in the pathogenesis of breast cancer (BC). YB-1 is another transcription factor that has been shown to promote stemness in BC cells. Methods: Western blotting, quantitative PCR, and siRNAs were used to query the regulatory relationships between YB-1, Sox2, and their downstream targets. Chromatin immunoprecipitation was used to detect YB-1 interactions at the Sox2 promoter. Mammosphere and soft agar assays were used to assess the phenotypic consequences of YB-1 knockdown. Results: Here, we report that YB-1 regulates Sox2. YB-1 was found to bind to the SOX2 promoter and down-regulate its expression in MCF7 and ZR751. The regulatory interaction between YB-1 and Sox2 was drastically different between the two phenotypically distinct cell subsets, purified based on their differential response to a Sox2 reporter. They are referred to as the reporter unresponsive (RU) cells and the reporter responsive (RR) cells. Upon siRNA knockdown of YB-1, RU cells showed an increase in Sox2 expression but no change in Sox2 reporter activity; in contrast, RR cells exhibited increased expression and reporter activity of Sox2. Correlating with these findings, YB-1 knockdown induced a differential response in the expression of genes known to be regulated by both Sox2 and YB-1 (e.g. CCND1 and ITGA6). For instance, in response to YB-1 knockdown, CCND1 and ITGA6 expression were decreased or unchanged in RU cells but paradoxically increased in RR cells. Compared to RU cells, RR cells were significantly more resistant to the suppression of mammosphere formation due to YB-1 knockdown. Importantly, mammospheres derived from parental MCF7 cells treated with YB-1 siRNA knockdown exhibited higher expression levels of SOX2 and its downstream targets. Conclusions: To conclude, in a subset of BC cells, namely RR cells, YB-1 regulates Sox2 to coordinately maintain stemness and tumorigenic properties. [ABSTRACT FROM AUTHOR]

Y-box binding protein-1 (YB-1) plays an important role in tumor progression and drug resistance. This study examined whether YB-1 is involved in the alteration of response to endocrine therapy in estrogen receptor (ER)-positive breast cancer cells. MCF7 cells that stably expressed YB-1 (MCF7-YB-1) and vector control cells (MCF7-vector) were established. These cells were used to analyze the expression of the factors related to ER and growth factor receptor signaling pathways and responses to antiestrogens (tamoxifen and fulvestrant) and estrogen responsive element (ERE) activity. The effect of knocking down endogenous YB-1 expression was tested in wild-type MCF7 cells. In addition, the expression of YB-1 and the factors related to ER and growth factor receptor signaling pathways were evaluated in clinical breast cancers treated with preoperative chemotherapy. The expression of HER2, AIB1, p-Erk, and c-Myc was increased in MCF7-YB-1 cells. In contrast, knocking down of YB-1 decreased the expression of these factors but increased the expression of ERα in wild-type MCF7 cells. Furthermore, sensitivity to antiestrogens was decreased in the MCF7-YB-1 in comparison to that in MCF7-vector cells. The introduction of YB-1 into MCF7 cells inhibited apoptosis and cell cycle arrest at G1 phase induced by antiestrogens. In MCF7-YB-1 cells, the expression levels of p-Erk and c-Myc were continuously upregulated when cells were treated with either tamoxifen or fulvestrant. The ERE activity was reduced in MCF7-YB-1 cells in comparison to MCF7-vector cells, and the ERE activity in MCF7-YB-1 cells was inhibited by fulvestrant at a lower concentration than that which inhibited the ERE activity in MCF7-vector cells. In ER-positive clinical breast cancers treated with preoperative chemotherapy, significantly more number of specimens that showed increased or positive YB-1 expression after chemotherapy was positive for HER2 expression. These data suggest that alteration of YB-1 may modify the crosstalk between the ER pathway and HER2 pathway in ER-positive breast cancer cells, and consequently, may alter the response to endocrine therapy in ER-positive breast cancer cells. [ABSTRACT FROM AUTHOR]

Abstract: YB-1 is a member of the cold shock domain family, with complex roles in DNA structure, gene transcription and translation. YB-1 promotes chromosomal instability, and mammary gland transgenic expression induces tumors with 100% penetrance. YB-1 is linked to poor prognosis in breast carcinoma and is a strong predictor of relapse and disease-specific survival. Survival is directly tied to the extent of local invasion and distal metastasis, processes dependent upon the activity of the membrane type I-matrix metalloproteinase, MT1-MMP. Non-invasive MCF-7 breast adenocarcinoma cells were transfected with YB-1/EGFP. YB-1 protein was detected in the invadopodia of cells with a migratory phenotype. There was increased expression of MT1-MMP protein concentrated at the leading edges of motile cells, which were highly invasive in collagen three-dimensional culture. The rates of MT1-MMP protein endocytosis and recycling to the cell surface were elevated in clones expressing higher levels of YB-1 protein. Control MCF-7 cells formed nonfatal, non-invasive, differentiated adenocarcinomas in vivo. MCF-7 cells expressing a twofold increase in YB-1 formed highly anaplastic tumors with local invasion, pulmonary metastases and high lethality. We conclude that YB-1 contributes to the development of an invasive, metastatic breast carcinoma phenotype by enhanced presentation of MT1-MMP at the sites of cellular invasion. [ABSTRACT FROM AUTHOR]

PIK3CA, which codes for the p110α catalytic subunit of phosphatidylinositol-3-kinase (PI3K), is implicated as an oncogene. Despite importance of PIK3CA in cancer, little is known about what drives up its expression in tumor cells. We recently characterized the PIK3CA promoter and reported that it is transcriptionally silenced by the tumor suppressor protein p53. In the present study, we demonstrate that PIK3CA can be induced by the oncogenic transcription factor Y-box binding protein-1 (YB-1). Three YB-1-responsive elements were identified on the PIK3CA promoter using chromatin immunoprecipitation and electrophoretic mobility shift assays. Interestingly, silencing YB-1 with siRNA in models of basal-like breast cancer decreased p110α protein levels regardless of whether PIK3CA was wild type, amplified or mutated. This decrease in p110α led to a reduction in PI3K activity and the downstream signaling primarily through p90 ribosomal S6 kinase and S6 ribosomal protein. Disruption in PIK3CA-dependent signaling suppressed cellular invasion correlative with loss of urokinase plasminogen activator (uPA). Similarly, silencing YB-1 suppressed invasion and uPA production however this was reversible through the introduction of constitutively active PIK3CA. In conclusion, YB-1 is the first reported oncogene to induce the expression of PIK3CA through transcriptional control of its promoter.Oncogene (2009) 28, 2406–2418; doi:10.1038/onc.2009.81; published online 11 May 2009 [ABSTRACT FROM AUTHOR]

Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer with a poor prognosis. Long noncoding RNAs (lncRNAs) play critical roles in human cancers. Krüppel-like Factor 5 (KLF5) is a key oncogenic transcription factor in BLBC. However, the underlying mechanism of mutual regulation between KLF5 and lncRNA remains largely unknown. Here, we demonstrate that lncRNA KPRT4 promotes BLBC cell proliferation in vitro and in vivo. Mechanistically, KLF5 directly binds to the promoter of KPRT4 to promote KPRT4 transcription. Reciprocally, KPRT4 recruits the YB-1 transcription factor to the KLF5 promoter by interacting with YB-1 at its 5' domain and forming an RNA-DNA-DNA triplex structure at its 3' domain, resulting in enhanced transcription of KLF5 and ultimately establishing a feedforward circuit to promote cell proliferation. Moreover, the antisense oligonucleotide (ASO)-based therapy targeting KPRT4 substantially attenuated tumor growth in vivo. Clinically, the expression levels of YB-1, KLF5 and KPRT4 are positively correlated in clinical breast specimens. Together, our data suggest that KPRT4 is a major molecule for BLBC progression and that the feedforward circuit between KLF5 and KPRT4 may represent a potential therapeutic target in BLBC. [ABSTRACT FROM AUTHOR]

Abstract: YB-1 is a protein involved in DNA repair, transcription, splicing, translation, and confers cisplatin resistance in several cancers. However, it is unknown which YB-1 activity is required for this resistance. To identify the mechanism(s) by which nuclear YB-1 confers cisplatin resistance, we generated several YB-1 mutants and tested their impact on resistance in the mammary tumor cell lines MCF7 and MDA-MB-231. Transfection of wild type YB-1 bestowed cisplatin resistance in such cells but a mutant YB-1 with a point mutation at position 175 (YB-1(E175A)) did not. A truncated YB-1(1–205) increased cisplatin resistance above the levels conferred by wild type YB-1. The truncated YB-1(1–205) has intact nuclease activities but could not separate a DNA duplex containing a Y-box sequence (activities associated with DNA repair). Moreover, this truncated YB-1(1–205) did not alter splicing of the adenovirus E1A pre-mRNA minigene as it had low binding affinity for several splicing factors. In contrast, the mutant YB-1(E175A) protein behaved like wild type YB-1 regarding all these activities but yet did not confer cisplatin resistance. Finally, transfection of mutant YB-1(E175A) had low impact on overall transcription. The wild type and truncated YB-1(1–205) induced important but different alterations in gene expression as revealed by microarray analyses. Our results indicate that the splicing and the nuclease activities associated with YB-1 have minor impact on cisplatin resistance. In contrast, the global expression profiles displayed by both wild type and truncated YB-1(1–205) revealed several chemoresistance signatures which differed depending on the genetic status of the breast cancer cell line used. [Copyright &y& Elsevier]

We have investigated protein kinase C (PKC) signaling, a putative differentiation-related and metastasis suppressor gene Cap43/NDRG1/Drg-1, and Y-box binding protein-1 (YB-1) to identify new molecular targeting for breast cancer. PKC is a family of serine/threonine kinases that is involved in the regulation of cell growth. We have demonstrated that PKC caused G1 arrest in a breast cancer cell line through a mechanism involving a PKC–ERK MAPK–JNK–Rb protein signaling pathway. We have also characterized a novel mechanism through which all- trans retinoic acid (ATRA) and antineoplaston, anticancer drug, caused cell growth inhibition in breast cancer cells through effects on intracellular pathways. ATRA decreased the expression of PKCα, as well as reduced ERK MAPK phosphorylation, and consequently caused G1 arrest. Antineoplaston caused the down-regulation of PKCα protein expression, resulting in inhibition of ERK MAPK phosphorylation, with resultant inhibition of Rb phosphorylation leading to G1 arrest. PKC signaling represents a promising target for development of novel therapeutic agents. Cap43 is known as N-myc downstream-regulated gene 1 (NDRG1). Treatment with estradiol (E2) significantly decreased the expression of Cap43 in ER-α-positive breast cancer cell lines. Co-administration of tamoxifen abrogated the E2-induced downregulation of Cap43 in ER-α-positive cell lines. These results suggested that Cap43 may hold the potential of being a molecular marker to determine the therapeutic efficacy of anti-estrogenic anticancer agents in breast cancer. YB-1 is a member of the cold shock domain protein family. The expression of nuclear YB-1 was correlated with HER2 positively in clinical specimens of human breast cancer. Immunostaining studies showed that nuclear YB-1 expression was an independent prognostic factor of overall survival. Expression of nuclear YB-1 played an essential role in acquirement of malignant characteristics through HER2-dependent pathways in breast cancer patients. PKC, Cap43 and YB-1 may be useful in new molecular-targeting diagnosis and therapeutics in breast cancer. [ABSTRACT FROM AUTHOR]

The epidermal growth factor receptor (EGFR) is linked to poor outcome in breast cancer, and resistance to hormonal therapy is often accompanied by activation of growth factor receptors. To investigate the mechanism(s) by which EGFR becomes activated in breast cancer, we screened a cDNA expression library for genes that mediate EGF-independent proliferation of human mammary epithelial cells (HMECs). We isolated the NSEP1 cDNA encoding Y-box-binding protein 1 (YB-1), a multifunctional transcriptional and translational regulator. This cDNA conferred growth factor independence to HMECs. YB-1-transduced cells overexpressed EGFR, but ErbB-2 (Her-2/neu) levels were unchanged. Moreover, EGFR was constitutively phosphorylated in the absence of exogenous ligand. In these cells, an EGFR-blocking antibody failed to inhibit proliferation, conditioned medium activity could not be detected, and the synthesis of EGFR ligands was reduced compared to parental cells. This suggests that EGFR is activated in a ligand-independent fashion. However, cell growth could be blocked with an ErbB kinase inhibitor, indicating that EGFR signaling plays a major role in YB-1-induced growth factor independence. Taken together, our results demonstrate that YB-1 overexpression can induce EGF independence in HMECs via activation of the EGFR pathway. This could represent one of the mechanisms by which YB-1 contributes to breast tumor aggressiveness.Oncogene (2005) 24, 3177-3186. doi:10.1038/sj.onc.1208504 Published online 21 February 2005 [ABSTRACT FROM AUTHOR]

Drug resistance is a common clinical problem in cancer treatment. The overexpression of P-glycoprotein appears to be closely associated with multi-drug resistance to anticancer agents. YB-1 binds to the Y-box in the promoter region of MDR1, which encodes P-glycoprotein. We evaluated the correlation between nuclear YB-1 and P-glycoprotein expression and other molecules, such as hormonal receptors, angiogenic factors, immune factors, and methalloprotease in 63 human breast cancers. Nuclear YB-1 expression had a significant correlation with P-glycoprotein and PgR expression, and also with CD68 grade, concerning accumulation of tumor associated macrophages. This series did not demonstrate P-glycoprotein and nuclear YB-1 expression might be one of the useful prognostic marker of breast cancer. [Copyright &y& Elsevier]