MKNK1 is a YB-1 target gene responsible for imparting trastuzumab resistance and can be blocked by RSK inhibition.

Trastuzumab (Herceptin) resistance is a major obstacle in the treatment of patients with HER2-positive breast cancers. We recently reported that the transcription factor Y-box binding protein-1 (YB-1) leads to acquisition of resistance to trastuzumab in a phosphorylation-dependent manner that relies on p90 ribosomal S6 kinase (RSK). To explore how this may occur we compared YB-1 target genes between trastuzumab-sensitive cells (BT474) and those with acquired resistance (HR5 and HR6) using genome-wide chromatin immunoprecipitation sequencing (ChIP-sequencing), which identified 1391 genes uniquely bound by YB-1 in the resistant cell lines. We then examined differences in protein expression and phosphorylation between these cell lines using the Kinexus Kinex antibody microarrays. Cross-referencing these two data sets identified the mitogen-activated protein kinase-interacting kinase (MNK) family as potentially being involved in acquired resistance downstream from YB-1. MNK1 and MNK2 were subsequently shown to be overexpressed in the resistant cell lines; however, only the former was a YB-1 target based on ChIP-PCR and small interfering RNA (siRNA) studies. Importantly, loss of MNK1 expression using siRNA enhanced sensitivity to trastuzumab. Further, MNK1 overexpression was sufficient to confer resistance to trastuzumab in cells that were previously sensitive. We then developed a de novo model of acquired resistance by exposing BT474 cells to trastuzumab for 60 days (BT474LT). Similar to the HR5/HR6 cells, the BT474LT cells had elevated MNK1 levels and were dependent on it for survival. In addition, we demonstrated that RSK phosphorylated MNK1, and that this phosphorylation was required for ability of MNK1 to mediate resistance to trastuzumab. Furthermore, inhibition of RSK with the small molecule BI-D1870 repressed the MNK1-mediated trastuzumab resistance. In conclusion, this unbiased integrated approach identified MNK1 as a player in mediating trastuzumab resistance as a consequence of YB-1 activation, and demonstrated RSK inhibition as a means to overcome recalcitrance to trastuzumab.

The expression of activated Y-box binding protein-1 serine 102 mediates trastuzumab resistance in breast cancer cells by increasing CD44+ cells.

The development of acquired resistance to trastuzumab remains a prevalent challenge in the treatment of patients whose tumors express human epidermal growth factor 2 (HER2). We previously reported that HER2 overexpressing breast cancers are dependent on Y-box binding protein-1 (YB-1) for growth and survival. As YB-1 is also linked to drug resistance in other types of cancer, we address its possible role in trastuzumab insensitivity. Employing an in vivo model of acquired resistance, we demonstrate that resistant cell lines have elevated levels of P-YB-1(S102) and its activating kinase P-RSK and these levels are sustained following trastuzumab treatment. Further, to demonstrate the importance of YB-1 in mediating drug resistance, the expression of the active mutant YB-1(S102D) rendered the BT474 cell line insensitive to trastuzumab. Questioning the role of tumor-initiating cells (TIC) and their ability to escape cancer therapies, we investigate YB-1's role in inducing the cancer stem cell marker CD44. Notably, the resistant cells express more CD44 mRNA and protein compared with BT474 cells, which correlated with increased mammosphere formation. Expression of YB-1(S102D) in the BT474 cells increase CD44 protein levels, resulting in enhanced mammosphere formation. Further, exposing BT474 cells to trastuzumab selected for a resistant sub-population enriched for CD44. Conversely, small intefering RNA inhibition of CD44 restored trastuzumab sensitivity in the resistant cell lines. Our findings provide insight on a novel mechanism employed by tumor cells to acquire the ability to escape the effects of trastuzumab and suggest that targeting YB-1 may overcome resistance by eliminating the unresponsive TIC population, rendering the cancer sensitive to therapy.

Effect of a prostaglandin EP4 receptor agonist on early fixation of hydroxyapatite/titanium composite- and titanium-coated rough-surfaced implants in ovariectomized rats.

The agonist of the prostaglandin EP4 receptor can increase bone density in osteoporosis. Using ovariectomized (OVX) and sham-operated (SO) rats, the effects of the EP4 receptor agonist, ONO-4819, and hydroxyapatite (HA) on implant-bone fixation in implants with a rough surface were investigated. Female Wistar rats (12 weeks old) were divided into either SO or bilateral OVX groups. Twenty four weeks later, either hydroxyapatite/titanium (HA/Ti) composite-coated or Ti-coated implants were implanted into the femora, and the animals were treated with either ONO-4819 or saline for 4 weeks. The fixation strength of the HA/Ti-coated implants was higher than that of the Ti-coated implants in the saline-treated OVX rats. In the OVX rats, ONO-4819 enhanced fixation of the rough Ti-coated implants to levels similar to that of HA/Ti-coated implants. These data suggest that a combination of treatment with an EP4 receptor agonist and a rough-surfaced implant might be useful in increasing the early fixation of cement-less arthroplasty, particularly in elderly patients with osteoporosis.

The transcriptional induction of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1.

PIK3CA, which codes for the p110alpha 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 p110alpha protein levels regardless of whether PIK3CA was wild type, amplified or mutated. This decrease in p110alpha 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.

Profiling YB-1 target genes uncovers a new mechanism for MET receptor regulation in normal and malignant human mammary cells.

Basal-like breast cancers (BLBCs) are aggressive tumors with high relapse rates and poor survival. We recently reported that >70% of primary BLBCs express the oncogenic transcription/translation factor Y-box binding protein-1 (YB-1) and silencing it with small interfering RNAs (siRNAs) attenuates the growth of BLBC cell lines. To understand the basis of these earlier findings, we profiled YB-1:DNA complexes by chromatin immunoprecipitation (ChIP)-on-chip. Several tumor growth-promoting genes such as MET, CD44, CD49f, WNT and NOTCH family members were identified. In addition, YB-1 and MET are coordinately expressed in BLBC cell lines, as well as in normal human mammary progenitor cells. MET was confirmed to be a YB-1 target through traditional ChIP and gel-shift assays. More specifically, YB-1 binds to -1018 bp on the MET promoter. Silencing YB-1 with siRNA decreased MET promoter activity, transcripts, as well as protein levels and signaling. Conversely, expressing wild-type YB-1 or a constitutively active mutant YB-1 (D102) increased MET expression. Finally, silencing YB-1 or MET attenuated anchorage-independent growth of BLBC cell lines. Together, these findings implicate MET as a target of YB-1 that work in concert to promote BLBC growth.

Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells.

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

Prostaglandin EP4 receptor agonist augments fixation of hydroxyapatite-coated implants in a rat model of osteoporosis.

The reduced stability of hydroxyapatite (HA)-coated implants in osteopenic conditions is considered to be a major problem. We therefore developed a model of a boosted cementless implantation in osteopenic rats.Twelve-week-old rats were either ovariectomised (OVX) or sham-operated (SO), and after 24 weeks plain or HA-coated implants were inserted. They were treated with either a prostaglandin EP4 receptor agonist (ONO-4819) or saline for one month. The EP4 agonist considerably improved the osteoporosis in the OVX group. Ultrastructural analysis and mechanical testing showed an improvement in the implant-bone attachment in the HA-coated implants, which was further enhanced by the EP4 agonist. Although the stability of the HA-coated implants in the saline-treated OVX rats was less than in the SO normal rats, the administration of the EP4 agonist significantly compensated for this shortage. Our results showed that the osteogenic effect of the EP4 agonist augmented the osteoconductivity of HA and significantly improved the stability of the implant-bone attachment in the osteoporotic rat model.

S-myotrophin promotes the hypertrophy of myotube as insulin-like growth factor-I does.

We have reported previously that a novel muscle cell growth factor, having a structure of a peptide with sugar chains, was successfully purified from porcine skeletal muscle. It was named s-myotrophin. To determine the role of s-myotrophin in skeletal muscle growth, the effect of s-myotrophin on primary cultured chick skeletal muscle cells (composed almost totally of multinucleated myotubes) was investigated by comparing s-myotrophin with Insulin-like growth factor-I (IGF-I). Both s-myotrophin and IGF-I significantly increased creatine kinase activity of the cultures; both substances gave similar responses. Intracellar protein content was also increased by the addition of these factors. The content of myosin and actin in s-myotrophin treated culture in the differentiation medium was significantly higher than that of the control (unstimulated). The content of those proteins in IGF-I treated culture was also higher than that of control, but the differences were not statistically significant. Immunoblot analysis confirmed that the amounts of myosin and actin in the myocytes were greatly increased by s-myotrophin stimulation and also by IGF-I stimulation. Morphological observations using an anti-desmin antibody staining procedure demonstrated that the size of both s-myotrophin and IGF-I treated myotubes was appreciably larger than that of control myotubes. These results suggest that s-myotrophin is a potent mediator of skeletal muscle cell hypertrophy thorough the accumulations of muscle structural proteins.

Lipolytic effect of BRL 35 135, a beta3 agonist, and its interaction with dietary lipids on the accumulation of fats in rat body.

The type of intaked fat and fat uptake mechanisms such as adrenergic-induced lipolysis affect patterns of fat accumulation in animal body. In this study, in vitro lipolytic effect of BRL 35135, a selectivebeta3 agonist, and its interaction with different dietary fats on fat accumulation in animal body (in vivo) were studied. For in vitro study, adipocytes isolated from epididymal fat were incubated with 10(-5) M -10(-9) M of either BRL 35135 or isoproterenol, a non-selectivebeta-agonist. In animal study, two groups of SD-rats, i.e., BRL35135-intaked (dosed at 0.5 mg/kg/day in diet) and control, were divided into 4 sub-groups and fed diets containing 12% of either beef tallow (BT), canola oil (CO), olive oil (OO) or safflower oil(SO) for 6 weeks. In vitro study showed that BRL 35135 was 10 times more potent than isoproterenol in increasing the lipolysis in rat adipocytes. In animal study, inclusion of BRL35135 reduced daily weight gain in CO and SO groups (P < 0.05). Abdominal fat weight in BRL35135-intaked group was significantly lower than control in all dietary sub-groups (CO, OO and SO) except BT (P < 0.05). In BT group, abdominal fat contained significantly higher amount of total saturated fatty acids (SFAs) compared to CO, OO or SO. It was concluded that, although BRL 35135 was very potent in increasing lipolysis in the isolated adipocytes of rat, its preventive effect on lipid accumulation in animal body through the lipolysis could be affected by the type of dietary fat and was lesser when rats fed fats rich in SFAs.