Levels A comprehensive review of the functions of YB-1 in cancer stemness, metastasis and drug resistance

Abstract

The Y Box binding protein 1 (YB-1) is a member of the highly conserved Cold Shock Domain protein family with multifunctional properties both in the cytoplasm and inside the nucleus. YB-1 is also involved in various cellular functions, including regulation of transcription, mRNA stability, and splicing. Recent studies have associated YB-1 with the regulation of the malignant phenotypes in several tumor types. In this review article, we provide an in-depth and expansive review of the literature pertaining to the multiple physiological functions of YB-1. We will also review the role of YB-1 in cancer development, progression, metastasis, and drug resistance in various malignancies, with more weight on literature published in the last decade. The methodology included querying databases PubMed, Embase, and Google Scholar for Y box binding protein 1, YB-1, YBX1, and Y-box-1.

Introduction

YB-1 is a multifunctional protein that took its name from its ability to bind a DNA sequence called the Y-box (5’-CTGATTGGC/TC/TAA-3’) located in the promoter region of many genes, and it is involved in their expression either directly or indirectly. Other less commonly used old names include p50 or DNA-binding protein dbpB [1,2]. While first recognized as a DNA transcription factor, several other functions were discovered and attributed to YB-1 over the years, ranging from DNA repair to pre-mRNA splicing, translation, and packaging [3]. YB-1 protein is localized primarily in the cytoplasm, but it is translocated to the nucleus when cells are exposed to UV radiation or treated with certain chemotherapies, such as cisplatin [4]. YB-1 is a 36 kDa protein consisting of three domains: the alanine/proline-rich N-terminal domain (A/P domain), the cold shock domain (CSD), and the large C-terminal domain (CTD) with alternating clusters of positively and negatively charged amino acid residues. CTD was previously believed to be a disordered protein [1]. A recent DNA structural analysis study revealed the CTD interacts with several residues in the CSD and adopts a rigid structure with the ability to recognize more DNA binding sites than previously thought. However, phosphorylation at S102 or deletion of CTD reduces DNA binding significantly. This proposes that YB-1 interaction with DNA and RNA can be regulated via S102 phosphorylation [5]. The nucleocytoplasmic distribution is probably facilitated by the presence of cytoplasmic retention site (CRS) at (267–293) and of a set nuclear localization signal (NLS): NLS-1 (149–156), NLS-2 (185–194), and NLS-3 (276–292). In a normal growing environment, CRS predominates over NSL, and YB-1 mainly remains in the cytoplasm. In contrast, proteasome 20S cleaves off the CRS-containing portion under stress and enables translocation to the nucleus [6]. Of note, CRS and NLS-3 overlap; Roeyen et al. showed that this peptide’s compartmentalization function depends on the phosphorylation status of tyrosine 281, with phosphorylation directing YB-1 to the nucleus [7]. See (Figure 1).

Figure 1.

Structure of YB-1 depicting three domains; Ala/Pro-rich N-terminal domain (1–50), cold shock domain (51–128) with phosphorylation site at serine 102, and C-terminal domain (129–324). The sites of cytoplasmic retention site (CRS), nuclear localization signal (NLS) and S20 proteasome cleavage are shown.

Cold Shock Domain and the evolutionary origin of YB-1

Cold shock domain (CSD) was discovered from the study of Escherichia coli to decreasing temperature (cold shock). Whereas synthesis of most proteins was suppressed, some proteins showed an increased expression, and among them was cold shock protein A (CspA) which consists of β-barrel consisting of five anti-parallel β-strands. Since, CSD-containing proteins have been detected in various prokaryotic and eukaryotic cells. Of CSD proteins in mammals, the YB-1 is among the most studied [8].

There are three family members of the Y box binding family; YBX1, 2, and 3. YBX1 gene is located on chromosome 1 at position 1p34 [9], YBX2 is present on chromosome 17p13. [10] and YBX3 is localized on chromosome 12p13 [11]. While YB-2 expression is limited to early development, YB-1 and YB-3 continue to be expressed beyond and may substitute each other’s given common CSD homology [12]. YB-1 has high to moderate expression in most tissues, e.g., gastrointestinal, lung, bone marrow, lymph nodes, skin, nervous system, thyroid gland, pancreas, kidney, breast, prostate, and ovaries [13]. We deduced a phylogenetic tree of the YBX proteins using the Clustal Omega program. Based on the protein sequence alignments, the YBX gene family seems to have originated from a single ancestor closely related to YBX2, which had split during evolution to generate both YBX1 and YBX3. A significant homology exists between human and mouse YBX genes, with YBX1 being almost 100% identical while YBX2 and YBX3 orthologs shared 88% and 87%, respectively. See Figure 2.

Figure 2. Evolutionary conservation of the YBX gene family.

(A) Alignment (Clustal Omega; https://www.ebi.ac.uk/Tools/msa/clustalo/) of the human and mouse YBX amino acid sequences. YBX1, NP_004550; Ybx1, NP_035862; YBX2, NP_057066; Ybx2, NP_001334563; YBX3, NP_001138898; Ybx3, NP_620817. (B) Deduced phylogenetic tree of the YBX proteins using the Clustal Omega program. Based on the protein sequence alignments, the YBX gene family seem to have originated from a single ancestor closely related to YBX2, which had split during evolution to generate both YBx1 and YBX3.(C) Percent Identity Matrix between the human and mouse YBX proteins. The human and mouse YBX1 are almost 100% identical compared to their YBX2 and YBX3 orthologs, 88% and 87%, respectively.

While phosphorylation at S102 is the most studied post translations modification of YB-1, other has been described. For example, in colon cancer cells HT29, phosphorylation of YB-1 at S165 promoted tumorigenesis via NF-κB activation [14]. And as described above, phosphorylation at tyrosine 281 determines nuclear and cytoplasmic localization of YB-1. Other predicted post-translational modifications, including ubiquitylation, acetylation, and methylation, are yet to be confirmed in in-vivo studies [15].

1. DNA-binding functions of YB-1

The role of YB-1 as a transcription factor has been first recognized when it was found to bind to the promotor of two genes: the major histocompatibility complex (MHC) class II [16], and the epidermal growth factor receptor (EGFR) enhancer gene [17]. This function was further confirmed when YB-1 was identified as a transcription regulator of a well-defined set of E2F1 regulated genes involved in cellular proliferation, including CDK1, CDC6, CCND1, MCM2, and TOP2A [18]. However, although YB-1 as a transcription factor is elucidated for specific genes, the story is far from complete. For example, in genome-wide chromatin immunoprecipitation (ChIP)-on-chip analysis of breast cancer cell lines, there were over 6000 potential target genes for YB-1 [19]. Furthermore, YB-1 plays a significant role in DNA repair, and this function stems from its increased affinity for DNA with an apurinic site, cisplatin-damaged DNA, or DNA with unpaired bases. Also, YB-1 has 3’–5’ exonuclease and endonuclease activity on ssDNA and dsDNA [20,21]. Transfection of human epidermoid KB cells, with YB-1-antisense oligo increased sensitivity to cisplatin, mitomycin C, and UV radiation but not vincristine, doxorubicin, camptothecin, or etoposide, suggesting YB-1 may protect cells from the cytotoxic effects of DNA cross-linking agents, possibly through a DNA repair process [22]. In MCF7 cells, YB-1 was found to bind and activate human endonuclease III (hNTH1) when cells were treated with UV light or cisplatin but not with mitomycin C or camptothecin, and inhibition of either YB-1 or hNTH1 sensitized cells to UV light or cisplatin [23]. Accordingly, various chemotherapy resistance genes are upregulated by YB-1, including multidrug resistance 1 (MDR1) gene, which codes for ATP-dependent drug export protein P-glycoprotein (P-gp) and major vault protein (MVP), also known as lung resistance-related protein (LRP), which is overexpressed in certain multidrug-resistant cancers. Y-box is a part of the promoters of these genes, and YB-1 is recognized on the cis-regulatory element of the MDR1 promoter and MVP promotor [24][25]. Moreover, expression of MDR1 correlates with nuclear localization of YB-1 and has been confirmed in multiple cancers, including breast, sarcoma [24,26,27]. However, in colon cancer, prostate cancer, gastric cancer, and pancreatic cancer, the MDR1 expression was independent of YB-1 [28–30]. This discrepancy could be explained by the need for another mediator protein, for example, AP-endonuclease 1 (APE1). APE1 is central in repairing oxidative base damage and DNA strand breaks and has a second activity as a transcription regulator. APE1 in the acetylated form stably interacts with YB-1 and enhances its binding to the Y-box element of the MDR1 gene at a higher affinity than YB-1. Downregulation of APE1 sensitizes cells to xenobiotics [31]. The relationship between YB-1 and MDR1 is complex and appears to be tissue and pathway-specific. Furthermore, YB-1 is a potent co-factor for DNA-repair enzyme Poly(ADP-ribose) polymerase 1 (PARP1), and it can reduce the efficiency of PARP1 inhibitors that are used in the treatment of several BRCA gene driven cancers [32].

2. RNA-binding functions of YB-1

The ability of YB-1 to bind to RNA was discovered in the 1970s when it was known as p50, which was long before its DNA binding ability was realized in late 1980s. YB-1 interacts with numerous mRNAs, and it is a potent cap-dependent mRNA stabilizer [33]. The phosphorylation status of YB-1 is a key regulator of YB-1 as ribonuclear protein. Non-phosphorylated YB-1 binds to translationally inactive messenger ribonucleoprotein particles (mRNPs) that inhibit internal ribosome-dependent mRNA translation. Once phosphorylated at S102, its mRNA binding ability weakens and permits the translation of mRNAs regulating cell proliferation and transformation. Also, phosphorylation of YB-1 allows its translocation to the nucleus with an increase in YB-1 related genes expression [34–38]. Evdokimova et al. showed that overexpression of YB-1 in multiple cancer cell lines directly activates cap-independent translation of mRNAs encoding for Snail1, Twist, HIF1a, Lef-1, Foxo3a, and HoxC5 [39]. Non-coding RNA (ncRNA) such as microRNA (miRNA), circular RNA (CircRNA), and long non-coding RNA (LncRNA) were previously dumbed as non-functional are now recognized as central regulators of cellular processes, and expression alteration is associated with several cancers [40]. YB-1 is involved in regulating microRNA, as shown in genome-wide analysis of YB-1-RNA interaction in glioblastoma multiforme where YB-1 binds pre-/pre-miR-29b-2 and regulates biogenesis by blocking interaction with Dicer [41]. Conversely, YB-1 is regulated by ncRNAs such miR137 in melanoma, where it was found to downregulate YB-1 [42], and certain LncRNA e.g., linc00637 serve as a decoy interfering with the YB-1 mediated translation of HIF1a [43]. Adding to the complexity of eukaryotic gene expression is that 95% of multiexon genes undergo alternative splicing [44]. In early studies of spliceosome structure, YB-1 was one of the first recognized pre-spliceosomes associated proteins (splicing factor) [45]. YB-1 was shown to interact with A/C-rich splicing enhancers (ACE) of CD44. CD44 exon v4 has three ACE elements, and expression of YB-1 correlated with higher expression of oncogenic exon v4 variant in CD44, a known stemness marker [46]. Conversely, the splicing activity of YB-1was found to be regulated by TLS/CHOP fusion oncoprotein, which results from a balanced translocation of an oncogenic protein named translocation liposarcoma protein (TLS, also called FUS) on q13 with C/EBP homologous protein (CHOP) on p11. In sarcoma cell lines transfected with the adenovirus E1A gene, YB-1 splicing function on E1A pre-mRNA was inhibited by TLS/CHOP fusion oncoprotein. The same group showed a similar finding with another translocation; Ewing’s sarcoma protein (EWS)/ Friend leukemia integration 1 transcription factor (Fli-1) [47,48]. Besides, treatment of breast adenocarcinoma cell line MCF-7 with camptothecin inhibited the interaction between Ewing’s sarcoma oncoprotein (EWS), an RNA polymerase II-associated factor, and YB-1 spliceosome, which resulted in skipping of the MDM2 gene, a negative regulator of p53 that encodes p53 ubiquitin ligase [49].

3. YB-1 role in cellular functions

3a. YB-1 role in proliferation and apoptosis

YB-1 plays a vital role in cancer cell proliferation, facilitating the cell cycle’s progression at the G1/S phase. In fact, the Y-box sequence is a part of the promotor of several cell cycle-regulated genes. In cell lines with constitutive overexpression of YB-1, nuclear accumulation of YB-1 correlated with increased cyclin A and cyclin B1 transcription and translation [50], while siRNA-induced inhibition of YB-1 caused a marked suppression of cell cycle-related gene CDC6 along with reduced expression of cyclin D1, CDK 1, and CDK2 and increased expression of cell cycle inhibitors p21 and p16 [51]. In colorectal, lung, and sarcoma cancer, YB-1 expression was associated with increased DNA topoisomerase II expression, which is essential for cell proliferation. However, since both YB-1 and topoisomerase II-a are activated by c-Myc, it is not possible to conclude if YB-1 can independently activate topoisomerase II-a [28,52,53]. Apoptosis is a piece of essential machinery by which the organism controls its population of cells. It is initiated either through an intrinsic process when a cell senses stress or as a response to an extrinsic stimulus. Defects in apoptosis may result in uncontrolled proliferation. YB-1 mediates the anti-apoptotic effect on some cancer cells through direct interaction with major apoptosis regulatory protein P53 [54]. In HER-2 overexpressing breast cancer cells, YB-1 inhibition resulted in apoptosis through downregulation of p-STAT3^S727[55], whereas in a multiple myeloma cell model, apoptosis was noted following inhibition of YB-1 [56]. The anti-apoptotic function of YB-1 was also observed in immortalized fibroblast cells, where YB-1 repressed p53-dependant proapoptotic genes Bax and Noxa [57]. The YB-1 mediated apoptosis inhibition is probably not universal and only of importance for a small number of cancers, likely due to the ability of cancer cells to switch off apoptotic programs through YB-1 independent mechanisms.

3b. YB-1 role in Epithelial-Mesenchymal Transformation and stemness

Epithelial-Mesenchymal Transition (EMT) is a cardinal process through which cancer cells can metastasize. Intracellular markers of EMT include loss of E-cadherin and tight junction protein ZO-1 and the acquisition of N-cadherin, zinc-finger Snail Family Transcriptional Repressor 1 SNAI1 (Snail), twist-related protein 1 (Twist1), SNAI2 (Slug), zinc-finger E-box-binding homeobox 1 (Zeb1), matrix metalloproteinase-2 (MMP-2) and vimentin (Vm). A study of several cancer cell lines, including skin, breast, and prostate, found that the E-box sequence in the promoter YB-1 is the target gene for Twist. Inhibition of either resulted in G1 arrest and reduced cellular growth. However, only re-expression of YB-1 and not Twist could recover cell growth [58]. In addition, enforced expression of YB-1 in multiple noninvasive epithelial cancer cells (breast, cervical, and prostate) resulted in increased expression EMT markers, mainly Snail, and transformation of cells into mesenchymal phenotype [39]. On the other hand, YB-1 role in stemness is most described in breast cancer. In basal-like breast cancer cell lines MDA-MB-231 and BT549, YB-1 downregulation was associated with a significant reduction in the mRNA expression level of CSC genes Oct-4, Nanog, and Sox2[59]. These findings were further confirmed by Yang et al., where YB-1 knockout significantly reduced tumorsphere formation and reduced the expression of stemness genes, including Oct-4, Nanog, ALDH1, and ABCG2 [60]. Besides, YB-1 correlation with resistance to UV radiation and various chemotherapeutic agents imply cancer stem cell properties.

3c. YB-1 role as a ligand in promoting angiogenesis and oncogenic activities

In addition to the plethora of intracellular functions discussed above. Extracellular roles of YB-1 proceed in the same theme of promoting oncogenesis. YB-1 can be secreted from cells in non-classical pathway mode; it binds to NOTCH-3 receptors and stimulates cellular proliferation and migration [61,62]. YB-1 present in the exosomes cargo of gastric cancer cells, and when the extract is exposed to umbilical vein endothelial cells, proliferation, migration, and invasion increased, as well as increased expression of multiple angiogenic factors [63]. YB-1 is actively secreted in melanoma cells, and increased secretion levels correlate with poor prognosis by promoting migration, invasion, and tumorigenicity [64] Angiogenesis is an integral part of tumorigenesis. Takahashi et al. showed high YB-1 is expressed in the endothelial bed of several cancers, including glioblastoma, gastroesophageal cancer, colon cancer, and lung cancer. In contrast, no or low YB-1 expression was observed in a normal angiogenic bed. In human umbilical vein endothelial cells model, siRNA-induced YB-1 inhibition resulted in cellular growth arrest. This hints that YB-1 may play a role in the tumor environment and in tumor-associated endothelial cells [65].

3. YB-1 regulation and signaling pathways

The protooncogene c-Myc is involved in regulating the expression of 15% of all genes, including genes involved in cell division, proliferation, and apoptosis. This is through the interaction with the E-box present in the promoter region of these genes [66]. c-Myc was shown to drive the expression of YB-1 in several cell lines [67,68]. Other regulators of YB-1 transcription include transcription factors Twist, and TGFβ mediator Smad2 [58,69]. Multiple signaling pathways affect YB-1 level and function. Signaling pathway mTOR that is known to drive cell growth and division is essential for YB-1 synthesis. Most cellular YB-1 mRNA is stored as mRNP, therefore inactive. However, when mTOR signaling is suppressed with inhibitor PP242 but not rapamycin, specific suppression of YB-1 mRNA synthesis and translation was observed [70]. And in a feed-forward loop, YB-1 downregulation suppresses mTOR and p-mTOR [55]. The infamous pathways PI3k/Akt and Ras/Raf/MAPK/ERK, in which an activating mutation is the driving mechanism for many cancers, co-regulate YB-1 by two main mechanisms. First, as mTOR is downstream of both pathways, and activation of either will increase YB-1 levels. Second, both can phosphorylate p90 ribosomal S6 kinase (RSK), which in turn phosphorylates YB-1 at S102, triggering translocation of pYB-1^S102 to the nucleus where it alters the expression of various genes (Figure 3) [9–11].

Figure 3.

Akt/PI3K, Ras/Raf/MAPK/ERK, mTOR, TGFβ/Smad2 all regulate YB-1. RSK phosphorylates YB-1 at S102 initiates translocation to the nucleus where YB-1 alters many genes related to oncogenesis. In the cytoplasm, YB-1 regulates mRNA translation and spliceosome

4. Role of YB-1 cancer progression and metastasis

4a. YB-1 and Breast Cancer

Breast cancer is the most common non-cutaneous malignancy in women worldwide. It is a heterogeneous disease and classified into four distinct molecular groups: luminal A and luminal B (expressing either estrogen receptor or progesterone receptor, or both), human epidermal growth factor receptor 2 (HER-2) expressing subtype, if it expresses neither a hormonal receptor nor HER-2, it is termed triple-negative breast cancer (TNBC). Though breast cancer curable in the early stages, once metastatic, it is considered incurable. Treatment modalities include surgery, radiation, endocrine therapy, HER-2 targeted therapy, conventional chemotherapy, and immunotherapy [71]. YB-1 is expressed in about 40% of breast cancer patients, with a higher expression exceeding 70% in basal-like breast cancers [72,73]. The earliest observation of YB-1 role in breast cancer was made by Bargou et al., who reported the treatment of drug-sensitive MCF-7 breast cancer cells with doxorubicin resulted in nuclear translocation of YB-1 with increased MDR-1 expression of P-gp and hence chemoresistance [26]. YB-1 expression is also augmented in several breast cancer cell lines when exposed to cisplatin, and over-expression of YB-1 was found to be sufficient in conferring resistance of breast cancer cell lines to cisplatin [21,23]. Bergman et al. found that in a transgenic mouse model, HA-tagged YB-1 provoked chromosomal instability that emerged from mitotic failure and chromosomal amplification. Interestingly, all multiparous transgenic mice developed invasive breast cancer with remarkably various histological types suggesting the role of YB-1 as a protooncogene [9]. The underlying mechanism was studied by Davies et al. they developed a model of pre-malignancy to characterize the role of YB-1 on tumorigenicity. Conditional expression of YB-1 altered several cell cycle-associated proteins with LIM kinase ½ (LIMK1/2) being most affected; the misexpression of LIMK1/2 was associated with cytokinesis failure with resultant centrosome amplification. Upon phosphorylation, YB-1 localized to the centrosome, it complexes with pericentrin and γ-tubulin and maintains the organelle structural integrity. Combined with YB-1-driven cyclin E expression, this led to slippage through the G1/S checkpoint and promoted proliferation of already genetically altered cells [74].

The prognostic utility of YB-1 is best illustrated by Habibi et al. who interrogated a cohort of 4049 breast cancer patients with 20 years of follow-up. YB-1 expression was detected in 41% by immunohistochemistry. In a subgroup that was considered low-risk, tumor tissue microarrays showed expression in 34% (437/1292). That was a strong predictor of relapse and disease-specific survival than estrogen receptor or HER-2 across all breast cancer subtypes [72]. Lasham et al., in a study of 375 tumors from a non-adjuvant treated breast cancer patient, found a strong association between YB-1 mRNA expression and higher-grade breast cancer and worse prognosis[18]. Janz et al. examined relapse risk in 83 patients who received postoperative breast cancer. They reported that patients with a high YB-1 expression 5-year relapse rate of 66% compared to no relapse in the low YB-1 expression group. Confirming the correlation between YB-1 expression and chemotherapy resistance [75]. Lastly, in a cohort of 27 breast cancer patients treated with paclitaxel, one-third of the patient had increased nuclear localization of YB-1 accompanied by increased P-gp expression. The other two-thirds had either no or discordant expression. The study suggests that YB-1 may be responsible for paclitaxel resistance in some breast cancer patients [76].

HER-2 amplification is observed in 15% of breast cancer, and it is a more aggressive subtype, and YB-1 is a possible mediator on the oncogenic potential of HER-2. In a cohort of 116 breast cancer patients, the correlation between hormone receptor (HR), HER-2 and YB-1 was examined. HER-2 positive tumors were frequently YB-1 positive, with HR−/HER2+ or HR+/HER2+ being YB-1 positive in 66.7% and 58.3%, respectively. Alternatively, in HER-2 negative cancer, YB-1 was expressed only in one-quarter of patients. In the TNBC subgroup, YB-1 expression did not predict response to chemotherapy [77]. Berquin et al. examined the relationship between YB-1, EGER (HER-1), and ErbB2 (HER-2) in immortalized non-cancer mammary epithelial cells H16N2, transduction of YB1 (H16-YB-1) allowed the cells to proliferate in serum-free media while H162 could not survive. EGFR was overexpressed but not ErbB2 (HER-2), and ErbB kinases inhibitor could block the effect. The study suggested that YB-1 overexpression can promote EGF independence via activation of the EGFR pathway [78]. However, on analysis of 389 breast cancer biopsies, YB-1 expression was positively associated with EGFR, Erb-2 (HER-2), and Ki67. The effect of YB-1 expression on EGFR and HER-2 was different between breast cancer cell lines, while it influenced both in MCF-7 cells; in MDA-MB-231, it only affected EGFR [79]. In HER-2 overexpressing breast cancer cells BT474-ml and Au565, YB-1 knockdown caused apoptosis through downregulation of p-STAT3^S727 but not p-STAT3^Y705 or total STAT3. By contrast, constitutively active p-STAT3 rescued YB-1 induced apoptosis [55]. These findings were confirmed in multiple Basal-like breast cancer cells cell lines [80].

Moreover, YB-1 is fundamental in maintaining stemness in breast cancer. It binds at the promoter of CSC genes CD44 and CD49f and promotes their expression. The upregulation of wild-type YB-1 or its activated form pYB-1^S102 in MDA-MB-231 and SUM 149 cell lines increased CD44 and CD49f levels. Treatment with paclitaxel increased YB-1 and CD44 expression, and when activated mutant YB-1^S102D was transduced into cancer cells, self-renewal, primary and secondary mammosphere formation 8-folds, but it was reversed when either CD44 or CD49f were suppressed [81]. Dhillon et al. further studied the effect of YB-1 on the development of resistance to trastuzumab in HER2 positive BT474 cell line; it was noted the exposure to trastuzumab increase phosphorylation of YB-1, increased stemness marker CD44 protein level, and tumorsphere ability. Inhibition of either CD44 or YB-1 restored sensitivity to trastuzumab [60]. WAVE3, an actin-cytoskeleton remodeling protein that plays essential roles in directing cell motility and cancer cell invasion, was shown to be required for translocation of YB-1 to the nucleus in TNBC cell lines MDA-MB-231 and BT549. YB-1 downregulation was also associated with a significant reduction in the mRNA expression level of CSC genes Oct-4, Nanog, and Sox2 [59]. Yang et al. in MDA-MB-231 and MCF-7 cancer stem cells, YB-1 knockout significantly reduced tumorsphere formation and reduced the expression of stemness genes, including Oct-4, Nanog, ALDH1, and ABCG2; however, the rescue of YB-1 did not promote tumorsphere formation nor the expression of stemness genes [82]. The Drosophila homolog of Dachshund (DACH1) is a cell fate factor involved in the organism development. Underexpression was described in breast cancer with significant expansion of the CSC population. Wu et al. showed that DACH1 suppressed EMT via repression of cytoplasmic YB-1 mediated translation of Snail by binding and blocking YB-1 [83]. In global gene expression of YB-1 silenced MDA-MB-231 cells, a differential expression of actin-binding protein coronin-1C (CORO1C) was noted, suggesting it as a potential mediator of YB-1 downstream effects. Lim et al. showed that CORO1C overexpression mediated increment in MDA-MB-231 cells migration and invasion was abrogated by YB-1 downregulation [84].

As mentioned above, phosphorylation of YB-1 at Ser102 is essential for nuclear translocation of YB-1 and subsequent activation of cellular proliferation. In breast cancer cells, Ser102Ala mutation disrupted PI3K/Akt-dependent phosphorylation of YB-1 and suppressed tumor cell growth [35]. The same effect is observed when p90 ribosomal S6 (RSK) was inhibited or deleted [34]. Therefore, it appears that in breast cancer cells, YB1 phosphorylation is mediated through at least two independent pathways; PI3k/Akt or Ras/Raf/MAPK/ERK/RSK. Tiwari et al. found that simultaneous targeting of MEK and PI3K could effectively block YB-1 phosphorylation at the S102 site, with concomitant inhibition of MDA-MB-231 cell proliferation [37]. A study of multiple breast cancer cell lines found that dual inhibition of Akt and ribosomal S6 kinase efficiently inhibited YB-1 mediated DNA repair of ionizing radiation-induced DNA damage rendering cells to be more radiosensitive [85]. While YB-1 is a substrate for PI3K/AK, it feeds into a positive feedback loop where YB-1 induces the expression of p110α catalytic subunit of PI3K (PIK3CA), which is a known driver of tumorigenesis [86]. Stope et al. reported an interplay between YB-1 and TGFβ pathway, in MCF-7 cell line, incubation with TGFβ1 led to increased phosphorylation of Smad2 which significantly amplified over-expression of YB-1 [69]. Finkbeiner et al. showed that in basal-like breast cancer cells (BLBCs) SUM149 and MDAMB-231 cells, MET is expressed at a significant level while not expressed at a considerable level in non-basal cell lines. They confirmed the affinity of YB-1 to MET promotor, where siRNA-mediated knockdown of YB-1 led to MET downregulation while overexpressing YB-1 increased MET expression [19]. Another way YB-1 enhances the metastatic ability of BLBCs is through modulation of cell adhesion and matrix proteins the matrix metalloproteinase-1 (MMP1) and β-catenin, the expression of both proteins was significantly lower in MDA-MB-231 YB-1 knockdown cells [87].

In MCF10A human mammary epithelial cancer cells, overexpression of YB-1 caused cells to take a cuboidal shape and transition to spindle-shaped fibroblast-like morphology. This was accompanied by loss of epithelial markers E-cadherin, ZO-1, and MUC1, and expression of mesenchymal markers N-cadherin, Snail, Twist1, fibronectin, Vm, and others. This phenomenon is referred to as Epithelial-to-Mesenchymal tarnation (EMT) [39]. When noninvasive MCF-7 breast adenocarcinoma cells were transfected with YB-1/GFP, YB-1 was detected in the invadopodia of migratory cells with increased expression of membrane-type I-matrix metalloproteinase (MT1-MMP). This protein is known to have a pivotal role in cell invasion and metastasis [88]. Ito et al. studied the function of YB-1 in response to endocrine therapy. In estrogen-dependent MCF7 cells, YB-1 overexpression upregulated HER-2, p-ERK, and c-Myc, while YB-1 knockdown caused the reverse and increased the expression of ERα. Moreover, sensitivity to anti-estrogen was decreased in YB-1 overexpressing MCF7 [89]. Yang et al. confirmed the negative regulatory effect of YB-1 on ERα in ERα expressing breast cancer cell lines MCF-7 and ZR-75–1. Moreover, they showed that YB-1 regulates stemness, and YB-1 knockdown cells expressed less of Oct-3/4, Nanog, ALDH1, and Sox2 [90]. Collectively, YB-1 is involved in all molecular subtypes of breast cancer promoting stemness, EMT, proliferation and chemotherapy resistance, with a stronger relationship with the more aggressive subtypes, HER-2 positive and TNBC. Either knocking down YB-1 or inhibiting its phosphorylation appears to reverse oncogenic properties.

4b. YB-1 and Prostate Cancer

Prostate cancer (PCa) is the most common male cancer and the second cause of cancer deaths in males in the United States [91]. About 90% of PCa are androgen-dependent, and treatment with androgen deprivation therapy either medically or surgically (bilateral orchidectomy) is usually effective in controlling the disease initially; however, PCa ultimately develops resistance and resumes growth, and turns into castration-resistant prostate cancer (CRPC). One of the mechanisms of CRPC is through the expression of androgen receptor variants. YB-1 plays an integral role in EMT in prostate cancer. As YB-1 expression increased with prostate cancer grade, there is an inverse relationship with E-cadherin. Forced expression of YB-1 induced mesenchymal morphology and upregulation of mesenchymal markers while silencing YB-1 reversed mesenchymal differentiation, cellular proliferation, migration, and invasion [92]. In a study of 332 prostate cancer patients followed over 15 years, YB-1 and (metastasis-associated protein 1 (MTA1) abundance but not mRNA levels were associated with a 3-fold increase in risk for requiring further hormonal treatment or radiation [93]. The prognostic value of YB-1 was best illustrated by tissue microarray and immunohistochemistry study of nearly seven thousand patients where the intensity of cytoplasmic YB-1 or nuclear YB-1 staining was significantly associated with advanced Gleason grade and poor prognosis. YB-1 as a prognostication biomarker performed best in the ERG-negative prostate cancer subgroup [94].

In human prostate cancer tissues, YB-1 nuclear expression correlates with prostate cancer Gleason grading, and in vivo murine studies of LNCaP prostate cancer cells revealed increased YB-1 expression after castration with upregulation of P-gp. P-gp upregulation resulted in a decrease in intracellular vinblastine, endogenous androgen, and dihydrotestosterone. Collectively these findings suggest that in prostate cancer progression, YB-1 expression and localization to the nucleus increase P-gp, which in turn lower intracellular androgen and may activate adaptive survival pathways in an androgen deprived milieu [95]. However, a different observation concerning the interaction between YB-1 androgen dependence was made by Shiota et al. in that YB-1 expression was associated with Gleason score and androgen receptor expression [96].

Over-expression of the androgen receptor plays a pivotal role in the pathogenesis of CRPC. Only in 20% of CRPC, the mechanism is AR gene amplification, and for the remaining 80%, it is at the transcription level [97]. YB-1 regulated AR expression at the transcriptional level and found that increased nuclear YB-1 signal significantly correlates with the Gleason score and AR expression in PCa tissues [96]. In a study of 79 Japanese patients with metastatic prostate cancer, single nucleotide polymorphism (SNP) in YB-1 (rs12030724), which may affect intronmedicated gene expression of YB-1, was associated with a lower risk of progression with a hazard ratio of 0.52 compared to wild type, this supports the evidence that YB-1 is a critical regulator of androgen receptor variant [98]. YB-1 is also implicated in the development of resistance to nonsteroidal antiandrogen enzalutamide through the activation of HER-2 [99], and upregulation of AR variant 7 expression. In CRPC cells, the sensitivity to enzalutamide was augmented when YB1 was knocked down [100].

Taxane chemotherapy in combination with ADT prolongs survival in patients with metastatic cancers; however, almost all progress. YB-1 is upregulated in CRPC, and treatment with docetaxel further upregulates YB-1 and is grants taxane resistance through TGF-β/Twist/YB-1 pathway and knockdown YB-1 sensitized prostate cancer cells to docetaxel [101]. Clusterin, a cytoprotective protein that confers broad-spectrum chemotherapy resistance and is upregulated in taxane-resistant prostate cancer cells. While clustrin overexpressing taxane-resistant cells suffered increase paclitaxel-induced apoptosis following YB-1 knockdown, the opposite was not observed as overexpression of YB-1 did not rescues cells from increased paclitaxel-induced apoptosis following clusterin knockdown. This study indicates that YB-1 transactivation of clusterin is a mediator of paclitaxel resistance in prostate cancer [102]. Mechanistically, taxane activates RAF-1, which actives ERK leading to activation of ribosomal S6 kinases (RSK) with resultant phosphorylation and activation of YB-1. In taxane-resistant prostate cancer cells, targeting RSK through siRNA or via a kinase inhibitor to indirectly downregulate YB-1 resulted in sensitization of cells to taxane [103].

4c. YB-1 and Lung Cancer and Mesothelioma

Lung cancer the most common cause of cancer death in the world [104]. It is mainly divided into non-small cell lung cancer NSCLC and small cell lung cancer SCLC. The NSCLC is further subdivided into squamous cell carcinoma, adenocarcinoma, and other less common subtypes. YB-1 expression was associated with poor prognosis in few studies, in a Japanese cohort of 196 patients with non-small cell lung cancer (NSCLC), 88 (44.9%) had positive YB-1 expression immunohistochemistry. Nuclear YB-1 expression was associated with lymph node metastasis and the disease’s stage and conferred poor prognosis [105]. A European study of 77 patients with NSCLC confirmed that nuclear YB-1 expression was correlated with poor prognosis but not associated with disease stage [106]. Hyogotani et al., in a study of 105 NCSLC patients, nuclear YB-1 expression significantly correlated with MVP and EGFR expression. In NSCLC cell lines A549 and Calu-3, downregulation of YB-1decreased EGFR in both, but MVP was reduced considerably in A549 [107]. A potential intermediatory regulator of YB-1/EGFR is lncRNA CAR intergenic 10 (CAR10), which is upregulated on air pollution exposure and smoky coal combustion generated carcinogen dibenz[a,h]antracene. CAR10 binds and stabilizes YB-1 and protects it from proteasomal degradation leading leads to upregulation of EGFR [108]. In NSCLC cell lines A549 and H1299, downregulation of YB-1 inhibited proliferation factors Cyclin D1 and CDC6 expression [109]. Another pathway through which YB-1 regulates tumor growth in lung adenocarcinoma is the MACC1/c-Met pathway. Metastasis associated with colon cancer-1 (MACC1) regulates c-Met signaling and induces EMT, tumor invasion, and metastasis in several cancers. Using the shRNA knock-down model of A549 and H1299, YB-1 downregulation was associated with markedly reduced MACC1 promotor activity, suppressed MACC1/c-Met pathway, and attenuated tumorigenesis in a xenograft mouse model [110]. In adenocarcinoma cell lines A549 and H1299, long non-coding RNA lnc00665 directly interacts with YB-1 and enhances its stability through inhibition of proteasomal degradation and stimulated nuclear translocation with the expression of angiogenesis factors and promotes capillary sprouting in-vitro and in-vivo; these findings support the role of YB-1 as a mediator of angiogenesis that could potentially be targeted [111]. Lou et al. when EGFR mutant lung cancer cell lines (PC-9) were treated with tyrosine kinase inhibitor (TKI) gefitinib, YB-1 was significantly upregulated through Akt pathway activation with MVP being a mediator. The study included a retrospective analysis of 85 patients; YB-1 was increased in tyrosine kinase inhibitor-resistant patients suggesting the YB-1/MVP axis may play a role in gefitinib resistance [112]. The role of YB-1 in EMT in lung cancer was illustrated by Ha et al. In NSCLC cell line A549, downregulation of YB-1 increased epithelial marker E-cadherin. At the same time, overexpression of YB-1 enhanced E-cadherin transcriptional repressors TGF-β1 via Akt activation, they also noted that TGF-β1 stimulation-induced YB-1 nuclear translocation and hence activation [113]. Long noncoding RNA MIR22HG is under-expressed in lung cancer, and that is associated with poor survival. Su et al. reported that it exerts its effect through the stabilization of YB1 [114].

Malignant pleural mesothelioma is an aggressive malignancy with very poor survival and limited treatment options. YB-1 is expressed in multiple mesothelioma cell lines, including MSTO-211H, VMC23, and REN cells. Knockdown of YB-1 results in a significant reduction of proliferation, migration, and invasion in all cell lines. Interestingly, when RNA sequence was done on the YB-1 knock-down version of the cells mentioned, each cell displayed a unique gene expression picture with different pathways being altered [115].

4d. YB1 and Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer globally, with the incidence rising as socioeconomic conditions improve in many parts of the world because of its relationship with obesity [104]. While localized disease and some oligometastatic disease can be cured surgically, the treatment of metastatic disease is non-surgical, including 5-FU based chemotherapy, EGFR inhibitor cetuximab, anti-angiogenesis agent bevacizumab, and others. However, almost all will develop resistance and progress, and metastatic disease is the usual cause of death [116]. YB-1 studies in CRC focus on it is role in mediating anticancer treatment resistance and its role in promoting metastasis. Shiraiwa et al. conducting immunohistochemistry studies of 124 CRC cancer patients, high-grade nuclear expression of YB-1 was detected in 62.9% of cases and found to be an independent predictor of poor prognosis [117]. EGFR is a tyrosine kinase that is overexpressed in approximately 80% of CRC patients, and it is an activator of the RAS/RAF/MAPK/ERK pathway. Activating RAS/RAF mutations is frequent in CRC and is a feature of some CRC cell lines, e.g., such as SW480, HT29, and HCT116. YB1 knockdown decreased cellular proliferation and migration decreased regardless of RAS/RAF status. Simultaneously, in wild type RAS/RAF cell line CaCo2, YB-1 knockdown showed a reduction in EGFR and proapoptotic alterations with reduced Bcl-2 and increased Bax-2. This suggests that role of YB1 might be dependent on the mutational status of cancer cells [118]. Maier et al. explored the effect of YB-1 on human CRC cells with different mutational status, cetuximab-resistant mutated KRAS(G13D) HCT116, and cetuximab-sensitive KRAS wild-type SW48 cells. They showed 5-FU significantly induced YB-1 phosphorylation at S102 in mutated KRAS(G13D) HCT116 but not KRAS wild-type SW48 cells, and that dual targeting of Akt and RSK significantly inhibited proliferation regardless of mutational status [119].

In a study of 10 colon cancer and 16 rectal cancer patients, YB-1 was overexpressed in almost all malignant tissues but not in the normal mucosa. In KM12C human colon cancer cells, YB-1 expression correlates strongly with DNA topoisomerase II-a and modestly with proliferating cell nuclear antigen (PCNA), but no correlation with MDR-1 was noted [28]. Stein et al. in a study of HCT116 colon cancer cells, showed that treatment with 5-FU, vincristine, or cisplatin all increased the expression of drug resistance protein MVP; furthermore, YB-1 overexpression in the same cell lines using YB-1 cDNA and in the absence of chemotherapy agents lead to increased basal expression of MVP. This points to YB-1 being a possible driver for non-MDR-related (5-FU) chemoresistance in colon cancer [25]. In the quest of identifying how YB-1 mediates resistance to chemotherapy, Tsofack et al. showed that YB-1 cDNA transfection conferred oxaliplatin resistance in two colorectal cell lines SW480 and HT29. Interrogating the Oncomine microarray database, a list of 24 potential interactor proteins with YB-1 were identified, and inhibition with siRNA of followed. Only downregulation of either NONO or RALY protein sensitized previously oxaliplatin resistant YB1 overexpressing cells. [120] Ghatak et al. found that treatment of SW948 with chemotherapy agent FOLFOX, induced expression of CD44v6 through PGE2/EP1-mTOR pathway resulted in increased YB-1 expression with subsequent maintenance of stemness. CD44 is a transmembrane glycoprotein and acts as a receptor for hyaluronan. CD44 has multiple variants depending on alternative splicing. CD44v6 is the only variant proven to promote gut adenomas [121]. Novel Myc-activated lncRNA MNX1-AS1 that is overexpressed in CRC and associated with poor prognosis was shown to mediate its effect through YB-1 stabilization inhibiting YB-1 ubiquitination and degradation [122].

4e. YB-1 and Melanoma

Melanoma is a cancer of melanocytes. While less common than non-melanoma skin cancers, it accounts for 75% of skin cancer deaths [123]. Schittek et al. studying different stages of melanoma from normal melanocyte to premalignant, malignant with horizontal spread, malignant with vertical and metastatic, the level of YB-1 was increased in a step-wise fashion. YB-1 down-regulation resulted in reduced cell proliferation [64,124]. YB-1 down-regulation with shRNA in the in-vitro study resulted in reduced proliferation, migration, and invasion and increased apoptosis and chemosensitivity. This was accompanied by the downregulation of genes involved in, e.g., MMP-2, bcl-2, Cyclin D1, p53, and p16 [124]. A follow-up study by the same group showed that proliferation was dependent upon MAPK and PI3K/Akt activation, and p53 overexpression increase YB-1 S102 phosphorylation, whereas NFkB signaling inhibited phosphorylation. They also noted that overexpression of YB-1 inhibited its translation efficiency, suggesting auto-regulatory feedback [125]. In contrast, cytoplasmic unphosphorylated YB-1 is associated with malignant melanoma cells acquiring EMT-like properties (expression of Snail, Twist, and Slug) [126]. Elevated YB-1 expression correlates with the expression of matrix collagenases in malignant melanoma cell lines, hence facilitating invasion and metastasis [123,127].

MicroRNAs are critical regulators in melanoma; of particular interest is miR-137 which low expression is associated with poor survival in metastatic melanoma. Among miR-137 targets are c-MET and YB1, and transfection of malignant melanoma cells with miR-137 mimics downregulated YB-1 expression [42]. In an exciting experiment examining the effect of cross-species microRNA, Yang et al. found that shrimp miR-S8 inhibited YB-1 through 3’UTR of YB-1 mRNA interference. Treating malignant melanoma cells with miR-S8 resulted in down-regulation of stemness genes Oct-3/4, Nanog, ALDH1, and ABCG2. A follow-up study investigating YB-1 knock out in stemness in in-vivo and in-vitro melanoma models showed that deletion of YB-1 was associated with reduced expression of stemness genes (FZD-1, p21, GLP-1, GINS1, and Notch2) and stemness transcription factors (Sox2, Sall2, Oct-4, POU3F2, OLIG1, and Bmi-1) and rescue of YB-1 resulted in the recovery of expression [82,128].

Another dimension of YB-1 studied in melanoma but not in other cancers is its use as part of constructing immunotherapy. Zaidi et al. conducted a study where they started by inducing melanoma in mice with B16 malignant melanoma cells harboring the HSVtk suicide gene, followed by treatment with anti-viral ganciclovir (target for HSVtk suicide gene), which resulted in significant tumor regression. However, the tumor did recur; at that point, treatment of these recurrent melanomas with vesicular stomatitis virus-cDNA (VSV-cDNA) library constructed from the same recurrent tumors resulted in cure in all mice. Further screening of the VSV-cDNA library identified IL-17, YB-1, Topoisomerase-IIα, cdc7 kinase, and BRAF as the main rejection antigens [129].

BRAF ^V600E/K is mutated in 50% of melanoma patients, and inhibitors vemurafenib and dabrafenib are approved treatments; however, treatment is short-lived, and almost all patients’ progress. Kosnopfel et al. found that in malignant melanoma cell lines, vemurafenib resistance is mediated by upregulation of p90 ribosomal S6 kinase (RSK), a MAPK/ERK downstream kinase that phosphorylates YB-1 S102. Inhibition of RSK using chemical inhibitors (BI-D1870 and LJH-685) significantly affected the viability of these cells [38]. Sechi et al. found that fisetin (dietary flavonoid) could suppress RSK dependent phosphorylation independent of its effect on ERK in in-vitro and in vivo melanoma models [123]. Jia et al. reported that the cathelicidin antimicrobial peptide LL-37 promotes YB-1 expression in malignant melanoma and squamous melanoma cell lines with enhanced viability migration and invasion. Macrophages and polymorphonuclear cells produce LL-37, and it has been implicated in the development of several cancer types, including ovarian, breast, prostate, and lung [130,131].

4f. YB-1 and Sarcoma

Sarcomas are rare malignant tumors of cells derived from the mesenchymal origin and include many subtypes. YB-1 plays various roles in different sarcoma subtypes. In a study of 69 untreated human osteosarcoma patients, a significant positive correlation between nuclear localization of YB-1 and expression of MDR1 was noted. However, it was not correlated with the histological grade, and prognostic value was not studied [24]. The same group found that in 54 biopsies of synovial sarcoma, nuclear localization is associated expression of MDR-1 and topoisomerase II-a, and it correlates with poor prognosis [52]. In osteosarcoma cell line Saos-2 YB-1 expression was c-Myc driven through the interaction of c-Myc with E-box promotor of YB-1 and tumor protein p73 was needed for this interaction [67]. Malignant transformation of several sarcomas involves the presence of fusion protein that result from balanced translocation. In sarcoma cell lines transfected with the adenovirus E1A gene, the splicing ability of YB-1 on E1A pre-mRNA was inhibited by fusion proteins TLS/CHOP and EWS/Fli-1 [47,48]. Osteosarcoma is the most common malignant bone cancer in under 20 years old. Several osteosarcoma cell lines showed reduced proliferation and inhibition of cyclin D1 and cyclin A when YB-1 was knocked down, and in 40 patients with osteosarcoma, YB-1 nuclear expression with strongly associated with worse outcome [132]. Xu et al., in a study of 168 osteosarcoma patients found an inverse relationship between miR-382 and metastases and survival. In several osteosarcomas cell lines, miR382 was inversely related to the expression of EMT and stemness markers, and this was at least partially mediated through YB-1[133]. Circular RNA Circ_0001658 via sponging miR382 was shown to upregulate YB1 in osteosarcoma cell lines, and expression of circ_0001658 correlated to some degree with early relapse in a cohort of osteosarcoma patients [134]. Somaskharan et al. showed that YB-1 localizes to stress granules, and within it, it directly binds 5’ UTR of G3BP1 and activates translation. G3BP1 is an RNP that is needed for stress granules assembly. G3BP1 downregulation in sarcoma xenograft prevents stress granules formation, tumor invasion and completely blocks lung metastasis in mouse models [135]. El-Naggar et al. found that class I histone deacetylase (HDAC) inhibitors MS-275, as increasing reactive oxygen species in sarcoma cells, block their capacity for invasion and metastasis. Mechanistically, MS-275 does so through acetylation of YB-1 CSD and blocks its binding with target mRNAs [136]. El-Naggar et al. also found the YB-1 binds to hypoxiainducible factor (HIF1A) mRNA induces its translation and observed YB-1-HIF1A axis dependent invasiveness and metastatic ability in sarcoma in vivo model [137].

4g. YB-1 and Ovarian Cancer

Ovarian cancer chemotherapy treatment usually includes a platinum cytotoxic agent; however, most would have a recurrence or progress due to resistance. The observation of nuclear YB-1 translocation when cells were exposed to cisplatin and associated resistance intrigued the question of YB-1 role in ovarian cancer. An early study in the Japanese population by Kamura et al. found nuclear YB-1 expression in 12/40 (30%) of patients with stage III ovarian cancer, but only 4/12 (33%) had overexpression of P-gp. Subset with nuclear YB-1 had a significantly worse prognosis with a median survival of 10.6 months compared to 25.7 months YB-1 negative group [138]. Yahata et al. showed that cisplatin-resistance human ovarian cancer cell lines had higher expression of nuclear YB-1 than cisplatin-sensitive counterparts. In a study of 35 Japanese patients with recurrent ovarian cancer patients with matched biopsies pre and post recurrence, ten of the paired cases turned from negative nuclear YB-1 to positive, and two cases showed a reverse pattern [139]. In a Canadian study of 192 ovarian cancer of various stages, including 135 stage III patients, there was no association between YB-1 expression with either survival or time to recurrence. Possible expiations for contradicting results could be smaller sampler size in earlier studies or differences in the YB-1 antibody used, or protocol [140].

YB-1 phosphorylation and nuclear translocation in ovarian cancer cells can be via two pathways. First, PI3K/Akt, and in taxane-resistant ovarian cancer cells (HeyA8 and SKOV3), the inhibition of focal adhesion kinase (FAK) decreased YB-1 phosphorylation in an AKT-dependent manner [141]. Second, lysophosphatidic acid receptor LPAR/Src/EGFR/p90RSK/YB-1. In the latter mechanism, YB1 upregulates EGFR amphiregulin (AREG) which in turn self-reinforce YB-1 phosphorylation [142]. In contrast to breast cancer, using ovarian cancer cell line SKOV-3 studied with high-density oligonucleotide array, the YB-1 inhibition upregulated MDR1 gene, and it did not alter MVP expression. Nevertheless, YB-1 inhibition was associated with an increase in protooncogene CXCR4 expression [143]. Torchairo et al. reported that YB-1 activation is necessary to sustain the fibronectin/ TGF-β1 dependent pro adhesive circuit in ovarian cancer. YB-1 slicing reduced the expression of fibronectin, TGF-β1, and EMT marker Snail [144]. Tailor et al. made a recent discovery of an azopodophyllotoxin molecule (SU056) that binds and inhibits YB-1. In human ovarian cell cancer, OVCAR8 and murine cell line ID8, SU056 inhibited cell proliferation, increased apoptosis, and in a murine syngeneic model, it inhibited tumor growth and metastasis with no observable liver toxicity. Moreover, it has a synergetic effect with paclitaxel and inhibited drug efflux [145].

4h. YB-1 and Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death globally. The main risk factors include hepatitis B and C virus infection and alcohol use [146]. In a study of 82 tissue samples of HCC, YB-1 expression was present in 73 samples (89%), and it was not significantly associated with poor prognosis. Follow-up data were available in 37 patients, and YB-1 expression did not correlate with disease-free survival [147]. Nevertheless, in vitro and in vivo models suggests that YB-1 is oncogenic and promotes HCC. Chao et al. using a hydrodynamic gene delivery technique to create a loss of function and gain of function mouse liver model found that YB-1 is involved in HCC cell proliferation, migration, and drug resistance. In HCC cell line HuH-7, YB-1 knockdown inhibited the signaling of the Wnt/β-catenin pathway and reduced the expression of EMT and CSC genes [148]. miR372, a microRNA-372 known to play a role in HCC, promotes proliferation of liver cancer cells via YB-1 phosphorylation through HULC looping dependent ERK1/2 and PTEN. Highly Upregulated in Liver Cancer (HULC) is LncRNA that is overexpressed in HCC [149]. Liao et al., when treated HuH-7 liver cancer cell model with multikinase inhibitor sorafenib, PI3K/Akt activity increased and subsequently increased phosphorylation of YB-1, suggesting that YB-1 might enhance resistance. Conversely, YB-1 knockdown caused a reduction in cell migration and invasiveness by suppressing EMT markers Snail, Twist1, Zeb1, MMP-2, and Vm [150].

Inflammatory mediator prostaglandin E₂ (PGE₂) may have a causal role in HCC. In human HCC cell lines Hep3B and Huh7, PGE₂ upregulated YB-1 via mTOR1 [151]. Liu et al. found that stress simulation by treating multiple human HCC cell lines with β-adrenergic agonist isoproterenol resulted in YB-1 phosphorylation at S102 via β-arrestin 1-dependent activation of PI3K/Akt pathway followed by translocation of pYB-1^S102 to nucleus with enhancement of EMT, while a knockdown of β-adrenergic receptors diminished these effects [146]. Tanaka et al. showed that in HCC cell line HepG2, compound 7-hydroxyindirubin strongly inhibited the actinomycin D induced nuclear translocation of YB-1 without significant cytotoxicity with resultant suppression of MDR1, MVP, EGFR, and CXCR4 genes, which are known to confer chemotherapy resistance [152]. A relationship between YB-1 and PD-1 was reported by Tao et al. In HCC patients, YB-1 expression was positively correlated to PD-L1, possibly via increasing expression as it binds to the PD-L1 promoter. YB-1 knockdown enhanced chemosensitivity and CD8⁺ recruitment in the tumor microenvironment in a syngeneic mouse model [153].

4i. YB-1 and Esophageal and Gastric Cancers

Gastric cancer (GC) is the third leading cause of cancer death worldwide [104]. Death is primarily due to metastatic disease. In a Japanese study of 98 advanced GC samples, 29.6% showed strong YB-1 expression on immunohistochemistry with a significant correlation with vascular invasion, liver metastasis, and lower disease-free survival, but no relationship with EMT phenotype or overall survival [154]. Wu et al. confirmed a correlation between vascular invasion and YB-1 in a cohort of 163 patients [155]. Xue et al. showed that YB-1 presence in exosomes cargo of gastric cancer cells and when the extract is exposed to umbilical vein endothelial cells, proliferation, migration, and invasion increased, and a rise in multiple angiogenic factors (VEGF, Ang-1, MMP-9, and IL-8) was noted with increased angiogenesis [63]. HER2-targeted antibody trastuzumab is an approved treatment modality against HER2-positive advanced GC. Shibata et al. found that nuclear YB-1 expression is significantly associated with HER-2 expression in tissues of 111 GC patients. In multiple human cancer cell lines, they showed that downregulation of YB-1 caused lower expression of HER-2 with increased sensitivity to lapatinib, a dual EGFR, and HER2 kinase inhibitor [156]. Guo et al. examined YB-1 expression in multiple human gastric cell lines, and the highest expression was in NUGC3. Downregulation of YB-1 was associated with significant migration reduction but did not affect cell invasion or the expression of EMT genes [157]. It appears that YB-1 in GC primarily affects tumorigenicity via its effect on tumor vascular bed promoting angiogenesis. In esophageal cancer, a study of 233 patients with esophageal squamous cell carcinoma showed that YB-1 was predominantly distributed in the cytoplasm, and increased expression was associated with higher recurrence and lower overall survival compared to low expression cohort [158].

4j. YB-1 and Neurological Cancers

Glioblastoma (GBM) is the most common primary brain tumor in adults and is associated with a 2-year survival of less than 25%, and in children, primary brain tumors are the second most common cancer. In forty-nine adult patients with GBM, the YB-1 expression showed a significant correlation with the grade of disease. In human glioblastoma cell line SF188, downregulation of YB-1 resulted in reduced expression of stemness markers Sox2, musashi-1, Bmi-1, and nestin, and reduced neurosphere growth [159]. Zheng et al. reported that in 108 patients with different glioma grades, mRNA and protein levels of YB-1 and nuclear localization of YB1 were found to be significantly higher in higher-grade tumors. A higher level of YB-1 was associated with a worse prognosis [160].

Kindlin-2, a protein that promotes tumorigenesis through regulation of intercellular and cell-matrix adhesion. The expression of kindlin-2 correlates with a higher grade of GBM and a worse prognosis. Kindlin-2 upregulates YB-1 and β-catenin and formed a transcriptional complex that enhanced EGFR expression. In human glioma cell line U-87 MG that inherently expresses kindlin-2, they found the downregulation of either YB-1 or β-catenin were detrimental for kindling driven proliferation, migration, invasion [161]. Observation of nuclear localization of YB-1 upon infection with adenovirus was exploited in engineering YB-1 dependent oncolytic virotherapy that showed promising results in-vivo and in-vitro models [162]. In a study of the molecular profile of pediatric GBM, YB1 was overexpressed in 38 of 53 (72%) patients. The research suggests two subtypes depending on Akt/Ras activity. The Akt/Ras active group had a worse prognosis, and, in this group, YB-1 was localized to the nucleus [163].

4k. YB-1 and Hematological malignancies

Diffuse large B-cell lymphoma (DLBCL) is the most common adult non-Hodgkin lymphoma. Nuclear expression of YB1 was positive in 42/68 (61.8%) of treatment naïve patients, and is correlated with clinical stage, bone marrow involvement, extra-nodal disease, and poor outcome. No observed correlation with P-gp was noted [164]. In human DLBCL cell lines, overexpressing YB-1 in innate intermediate YB-1 expressor SUDHL-10 cells and downregulation of YB-1 in intrinsic YB-1 over-expressor cell line OCI-Ly10 was studied. YB-1 significantly increased cyclin D1 and cyclin E, accelerating G1/s transition and proliferation. Also, YB1 increased the adhesion of DLBCL cells to fibronectin and conferred cell adhesion-mediated drug resistance to mitoxantrone, and upon silencing of YB-1, sensitivity was restored [165].

Multiple myeloma (MM) is a neoplasm originating from plasma cells. While normal plasma cells in the bone marrow do not express YB1, MM cells from 21/22 (95%) of patients’ samples and almost all MM cell lines strongly express YB-1. Studying MM cell line INA-6, siRNA-mediated suppression of YB-1 led to growth arrest and apoptosis, while transient over-expression led to resistance to doxorubicin-induced apoptosis [56]. Cobbold et al. found that in patients with MM and in MM cell lines that harbor C to T mutation in 5’ untranslated region of c-Myc binds more strongly to YB-1 and polypyrimidine tract-bind protein (PTB-1) compared to unmutated c-Myc. Lowering either PTB-1 or YB-1 levels decreased c-myc expression and inhibited cell proliferation of MM cell lines, suggesting that mutated c-Myc expression is mediated partly through YB-1 [166]. Bommert et al. reported that YB-1 translation regulation of c-Myc in YB-1 is independent of the mutational status of c-Myc and showed that in MM cell lines, c-Myc knockdown results in lower YB-1 levels in a feed-forward feedback loop [143].

4l. YB-1 and Neuroblastoma

Neuroblastoma is a childhood tumor that originates from the neural crest with good overall survival except for high-risk patients where the prognosis is poor. Wachowiak et al. detected high expression of YB-1 in 35 of 37 neuroblastoma cases; however, no correlation of YB-1 with stage or survival was observed [167]. Wang et al. reported that YB-1 knockdown of SH-SY5Y neuroblastoma cell line resulted in a significant reduction in cell proliferation and increased apoptosis in vitro and in vivo xenograft. A follow-up study reported that silencing of YB-1 decreased proliferation, invasion, metastasis, and enhanced cisplatin sensitivity via NF-κB signaling [168,169].

4m. YB-1 and other cancers

Bladder cancer is the second most common genitourinary malignancy. Shiota et al. demonstrated that in bladder cancer cell lines (TCCsup, UMUC3, T24, and KK47), Twist regulated YB-1 expression and enhanced cell growth, invasion, and resistance cisplatin and doxorubicin but not to 5-FU [170]. Transcriptome study of 75 bladder cancer patients showed YB-1 expression was associated with more advanced stage and lower survival [171]. Expression of YB-1 is also enhanced in clear cell renal cell carcinoma and related to an advanced stage, resistance to sunitinib [172].

Pancreatic adenocarcinoma is one of the most aggressive cancers, with a 5-year survival of around 5%. Shinkai et al. reported a strong correction between nuclear YB-1 expression and poor prognosis in 69 patients. In studying several human pancreatic cell lines, they showed that knockdown of YB-1 decreased cell proliferation, invasion, and metastasis in vitro and in vivo [173]. Hypoxia-induced lncRNA-BX111 is overexpressed in pancreatic cancer and correlates with increased proliferation and invasion in pancreatic cancer. It exerts its effect via activation of EMT regulator Zeb1, which in turn increases YB-1 [174]. Lu et al. reported that YB-1 was suppressed by miR-216a via direct binding to YB-1 3’-untranslated regions and showed an inverse relationship between levels of YB-1 and mir-216a in various human pancreatic cell lines [175]. In a study of 365 head and neck squamous cell carcinoma patients. YB-1 expressed was observed in high-grade HNSCC and was associated with a worse prognosis [176]. In 59 cervical cancer patients, YB-1 expression was higher than cervical intraepithelial neoplasia, and the level correlated to the risk of relapse following chemoradiation [177].

5. YB-1 as a potential biomarker

There has been a growing interest in liquid biopsies over the past decades. They may have a role in screening, early diagnosis of recurrence, and identifying response to treatment [178].Tacke et al. measured a secreted 18 kD fragment YB-1 (YB-1/p18) in human plasma by immunoblot. None of the 33 healthy volunteers had a positive result, while it was positive in 11/25 hepatocellular carcinoma patients and 16/20 of patients with advanced cancerous [179]. A follow-up study revealed that circulating YB-1/p18 was highly prevalent at 78% of many malignancies, including lung, breast, and hematologic. However, YB-1/p18 detection did not predict survival, and concerning level change in response to therapy, it failed to perform better compared to commonly available tumor markers [180].

6. Conclusions and perspectives

YB-1 is a part of an orchestra, and it orchestrates a myriad of cellular functions. It resides in the cytoplasm, where it interacts with coding and non-coding RNA to regulate translation and splicing globally. Upon phosphorylation, it shuttles into the nucleus to alter the transcription of several genes and takes part in DNA repair. YB-1 is overexpressed in most cancers, and it is a driver of oncogenesis stimulating cellular proliferation, exerting anti-apoptotic effects, enhancing EMT, advancing invasion, maintaining stemness, and augmenting resistance to various modalities of cancer treatment (see Table 1). Nonetheless, these effects are not generalized and appear to be cancer and tissue-specific. YB-1 expression correlates with advanced stage and worse prognosis in some cancers, but again, it is not universal. The discrepancy in YB-1 role among different cancers and between various phenotypes of the same cancer could be due to a variety of possibilities, including coexisting alteration in cytoplasmic regulators of YB-1 such miRNAs and LncRNAs that were shown to either suppress or stabilize YB-1, whether total YB-1 or pYB-1^S102 are increased, and the presence of an activating mutation in PI3K/Akt, MAPK, or mTOR pathways. What is promising is the sustained observation of the anti-oncogenic effect with YB-1 knockdown, with multiple studies confirming such findings in vivo and in vitro. To date, there is only one small molecule, SU056, that potently suppresses Y box binding protein1 (YB-1). SU056 is an azopodophyllotoxin, which biophysically binds to YB-1 with proven efficacy in ovarian cancer syngeneic model is encouraging [145]. Whereas few substances like grape seed procyanidin and anti-microbial peptide LL-37 were shown to reduce YB-1 level, the direct effect was not confirmed [130]. Further studies to identify to improve SU056 specificity to YB-1 and identify other compounds specific to YB-1 are needed. An alternative way to tackle YB-1 mediated effects is by inhibiting its phosphorylation by simultaneously targeting RSK and AKT, an approach that showed promising results in breast cancer and colon cancer [85][119]. The relationship between YB-1 and PD-L1 in hepatocellular cancer is noteworthy, and another avenue that is merit exploring in other cancer, YB-1, may mediate immune evasion and resistance to immune checkpoint inhibitors, and studies investigating this relationship are needed[153]. The ability to measure YB-1 fragments in blood might be helpful. While it did not predict survival and it did not perform better than established tumor markers, it ought to be noted that these observations were not studied in the context of YB-1 directed therapy, and cancers treated might have resistance to cancer treatment prior to treatment. Studies exploring optimization of measurement of YB1 fragments or detection of cDNA are needed to guide selection for treatment and to monitor response.

Table 1.

Summary of roles of YB-1 in cancer

Correlation with poor prognosis	Breast cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, head and neck cancers, melanoma, ovarian cancer, sarcoma, glioblastoma, neuroblastoma, multiple myeloma, and diffuse large B cell lymphoma
Role in proliferation	Breast cancer, prostate cancer, lung cancer, colon cancer, melanoma, ovarian cancer, sarcoma, hepatocellular carcinoma, gastric cancer, pancreatic cancer, bladder cancer, glioblastoma, mesothelioma, neuroblastoma, multiple myeloma, and diffuse large B cell lymphoma
Role in stemness	Breast cancer, colon cancer, melanoma, sarcoma, hepatocellular carcinoma, glioblastoma
Role in EMT	Breast cancer, prostate cancer, lung cancer, colon cancer, ovarian cancer, sarcoma, hepatocellular carcinoma, gastric cancer melanoma, bladder cancer, pancreatic cancer, and neuroblastoma
Role as anti-apoptotic	Breast cancer, colon cancer, melanoma, multiple myeloma, and neuroblastoma
Role in drug resistance	Breast cancer, prostate cancer, lung cancer, colon cancer, ovarian cancer, neuroblastoma, bladder cancer and diffuse large B cell lymphoma

Other possible novel mechanisms to target YB-1 include the use of siRNA. With the success of this approach evidenced by FDA approval of lipid nanoparticle to deliver siRNA to the human liver in 2018, one would expect an increased uptake of this technology, and using YB-1 as one of the targets is worth pursuing [181]. And while the COVID-19 pandemic devastated humanity and changed the world, one benefit is the progress made in utilizing mRNA. This may hasten the development of various cancer vaccines. Interestingly, YB-1 as an immunological target and its efficacy as a vaccine were reported in neuroblastoma and melanoma in the past two decades [182,183]. The YB-1 tale is far from over. Its involvement in the aggressive phenotype of various cancers and the plethora of research demonstrating its carcinogenesis provides a strong foundation to pursue the quest of finding a YB-1 directed treatment that will have the potential to be an invaluable tissue-agnostic cancer therapy.