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Correct spatial and temporal control of cell proliferation is of fundamental importance for tissue homeostasis. Its deregulation has been associated with several pathological conditions. In common with almost every aspect of plant and animal biology, cell proliferation is dominated by day-night rhythms generated by the circadian clock. However, our understanding of the crosstalk between the core clock and cell cycle control mechanisms remains incomplete. In this study, using zebrafish as a vertebrate model system, we show that the nuclear localization of the Y-box binding protein 1 (YB-1), a regulator of cyclin expression and a hallmark of certain cancers, is robustly regulated by the circadian clock. We implicate clock-controlled changes in YB-1 SUMOylation as one of the mechanisms regulating its periodic nuclear entry at the beginning of the light phase. Furthermore, we demonstrate that YB-1 nuclear protein is able to downregulate cyclin A2 mRNA expression in zebrafish via its direct interaction with the cyclin A2 promoter. Thus, by acting as a direct target of cyclic posttranslational regulatory mechanisms, YB-1 serves as one bridge between the circadian clock and its cell cycle control.

DNp63a , a critical pro-proliferative factor and a marker of epidermal stemness, associates with the Y-box binding protein 1 (YB-1), a member of the cold shock protein family. YB-1 is an oncogenic, nucleic acid binding protein with pleiotropic function. Mainly localized in the cytoplasm, YB-1 is involved in alternative splicing, regulation of transcription and translation. Here we show that DNp63a induces the accumulation of post-translationally modified forms of YB-1 in the nucleus.Sumoylation and ubiquitination are both involved in this phenomenon. Silencing of YB-1 in differentiating HaCaT kertinocytes induces cell cycle arrest. YB-1 knockdown cells show early decline of cyclin D1, increase of p21WAF and reduced expression of the envelop precursor protein, involucrin. In proliferating HaCaT and squamous carcinoma cells, YB-1 triggers cell detachment and apoptosis suggesting that the functional interaction between DNp63a and YB-1 cooperate to support survival and proliferation of basal and transformed keratinocytes.

The p63 protein is a member of p53 transcription factor family. The TP63 gene encodes isoforms that contain (TA) or lack (DN) a transactivation domain. The p63 protein plays an essential role for the development of skin and other epidermal derivatives. The ΔNp63α isoform is mainly expressed in the basal layer of the squamous epithelia and is restricted to progenitor cells with proliferative potential. We demonstrate that ΔNp63α can physically and functionally interact with the Y-box-binding protein 1 (YB-1).YB-1 belongs to the cold-shock domain protein superfamily and performs a wide variety of cellular functions, including transcriptional and translational regulation of proteins involved in the proliferation, survival and cellular differentiation. Inasmuch, as the level of YB-1 drastically increases in tumor cells, this protein is considered to be one of the most indicative markers of malignant tumors. The YB-1 C-terminal region contains a number of distinct sites that can be targeted for post-translational modifications such as phosphorylation, acetylation and ubiquitination. We have observed that expression of ΔNp63α, but not ΔNp63γ, causes accumulation of YB-1 high molecular weight forms in the nuclear compartment and we provide evidence that these are ubiquitinated YB-1 forms. The functional role of YB-1 ubiquitination by ΔNp63α is currently under investigation.

Y-box binding protein 1 (YBX-1 or YB-1) is an oncoprotein that promotes replicative immortality, tumor cell invasion and metastasis. The increase in the abundance of YB-1 in the cell or YB-1 translocation from the cytoplasm to the nucleus is characteristic of malignant cell growth. We have previously reported that ΔNp63α, a transcription factor that is known to play a pivotal role in keratinocyte proliferation and differentiation, promotes YB-1 nuclear accumulation. Here, we show that YB-1 is highly expressed in proliferating keratinocytes and is down-regulated during keratinocyte differentiation. ΔNp63α reduces YB-1 protein turnover and leads to accumulation of ubiquitin-conjugated YB-1 into the nucleus. Reduction of YB-1 protein level, following treatment with a DNA-damaging agent, is inhibited by ΔNp63α suggesting that YB-1 and ΔNp63α interplay can support keratinocyte proliferation and protect cells from apoptosis under genotoxic stress.

The Y-box binding protein 1 (YB-1) belongs to the cold-shock domain protein superfamily, one of the most evolutionarily conserved nucleic acid-binding proteins currently known. YB-1 performs a wide variety of cellular functions, including transcriptional and translational regulation, DNA repair, drug resistance, and stress responses to extracellular signals. Inasmuch as the level of YB-1 drastically increases in tumor cells, this protein is considered to be one of the most indicative markers of malignant tumors. Here, we present evidence that ΔNp63α, the predominant p63 protein isoform in squamous epithelia and YB-1, can physically interact. Into the nucleus, ΔNp63α and YB-1 cooperate in PI3KCA gene promoter activation. Moreover, ΔNp63α promotes YB-1 nuclear accumulation thereby reducing the amount of YB-1 bound to its target transcripts such as that encoding the SNAIL1 protein. Accordingly, ΔNp63α enforced expression was associated with a reduction of the level of SNAIL1, a potent inducer of epithelial to mesenchymal transition. Furthermore, ΔNp63α depletion causes morphological change and enhanced formation of actin stress fibers in squamous cancer cells. Mechanistic studies indicate that ΔNp63α affects cell movement and can reverse the increase of cell motility induced by YB-1 overexpression. These data thus suggest that ΔNp63α provides inhibitory signals for cell motility. Deficiency of ΔNp63α gene expression promotes cell mobilization, at least partially, through a YB-1-dependent mechanism.

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

Stearoyl-Coenzyme A desaturase 1 (SCD1) belongs to the fatty acid family of desaturases. In lactating ruminants, the SCD1 protein is highly expressed in the mammary gland and is relevant for the fatty acid composition of milk and dairy products. Bovine mammary epithelial cells (BME-UV1), cultured in vitro, have been proposed as a model to reproduce the biology of the mammary gland. The present study was designed to investigate the responsiveness of bovine SCD1 promoter to serum, insulin, oleic acid, and NFY transcription factor in BME-UV1 cells. A luciferase-based reporter assay was used to monitor the transcriptional activity of the SCD1 promoter region in BME-UV1 cells treated or not with insulin and/or oleic acid. The level of endogenous SCD1 mRNA was evaluated by Real time PCR. Insulin (20 ng/mL) induced a 2.0 to 2.5-fold increase of SCD1 promoter activity. Additionally, the effect of insulin was inhibited by oleic acid, serum components, and NFY enforced expression. Serum and NFY showed no synergistic or additive effect on SCD1 promoter activity suggesting that they repress SCD1 transcription through the same responsive element.