Role of Y-box binding protein-1 (YB-1) in breast cancer

The Y-box binding protein-1 (YB-1) is a multifunctional protein with roles in transcription, translation, DNA repair, and a recently identified function as an extracellular mitogen. YB-1 is over-expressed in various malignancies including breast carcinoma. Previous work from our laboratory has shown that YB-1 is expressed in approximately 40% of invasive breast carcinomas, and its expression correlates with relapse and poor survival. Further, the oncogenic potential of YB-1 has been demonstrated in breast cancer. In the studies presented in this thesis, we sought to understand the contribution of YB-1 as an oncogenic transcription factor to breast cancer. We focused our studies on the basal-like breast carcinoma (BLBC) and the human epidermal growth factor receptor 2 (HER2) over-expressing breast cancers, as patients with these subtypes suffer the worst prognosis. Using BLBC cell lines, we demonstrated that YB-1 induces expression of MET and PIK3CA to promote anchorage-independent growth and invasion respectively. These studies further identified YB-1 as a potential therapeutic target in BLBC. We then directed our focus to the HER2 over-expressing breast cancers. Although the development of trastuzumab (Herceptin®), a targeted therapy against HER2, has provided a substantial advance in the care of affected patients, resistance remains a prevailing challenge. We identified a novel mechanism by which signalling proteins, mitogen activated protein kinase interacting kinase (MNK) and p90 ribosomal S6 kinase (RSK), interact to increase phosphorylation of YB-1. In turn, phosphorylation of YB-1 promotes its nuclear translocation where it regulates transcription of genes involved in trastuzumab resistance. These results further suggest YB-1 as a therapeutic target to improve outcome for women with trastuzumab refractory disease. As a whole, the studies outlined in this thesis have contributed to our understanding of breast cancer pathogenesis and have identified novel aspects of YB-1 function in BLBC and in HER2 over-expressing breast carcinomas.Medicine, Faculty ofMedicine, Department ofExperimental Medicine, Division ofGraduat

Physical and functional interaction of p53 and p110α and implications in ovarian carcinogenesis

In approximately 40% of ovarian cancers, PIK3CA, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K) is amplified. This amplification correlates with increased PIK3CA transcription, p110α protein expression, and PI3K activity. Moreover, PIK3CA is implicated as an oncogene in ovarian cancers. Another common mutation in ovarian cancer leads to loss of p53 function. Alterations to p53 are known to be involved in tumour development and progression. Previous studies have shown that in benign cells, the p53 and PI3K pathways are connected through the regulation of PTEN by p53. Therefore, in the presence of p53, PTEN levels increase and exert their effect through decreasing P-AKT levels, and thereby pro-survival activities. In addition, it has been suggested that in cancer cells, p53 down-regulates the pro-survival pathway, independent of PTEN, by negatively regulating p110α levels. However, it has not been shown whether the p53 effect on p110α levels is direct or indirect and whether this interaction exists in benign cells. Our studies show, for the first time, a direct relationship between p53 and PI3K pathways. We used temperature sensitive cells, in which p53 function was regulated through the shift in temperature. We showed that p53 negatively regulates PIK3CA transcript and p110α levels through direct binding to the PIK3CA promoter. Moreover, we determined that the regulation of p110α levels by p53 is also present in ovarian cancer cells where overexpression of p53 significantly reduced p110α levels. In addition, for the first time, we identified two alternate promoters (promoter la and promoter lb) upstream of two alternate first exons (exonla and exonlb) that transcribe into two alternate transcripts with different 5' untranslated regions (5' UTRs). Our results determined direct binding of p53 to PIK3CA promoter la, and studies to determine whether this direct binding is responsible for the suppression of the promoter and the down-regulation of PIK3CA transcript and p110α levels are currently in progress. Our studies suggest that the loss of p53 in ovarian cancers may result in loss of transcription suppression of PIK3CA and therefore increased p110α levels and PI3K activity, which may in turn lead to increase in proliferation and resistance to apoptosis.Medicine, Faculty ofObstetrics and Gynaecology, Department ofGraduat

THE TRANSCRIPTION FACTOR Y-BOX BINDING PROTEIN-1 (YB-1) INDUCES EXPRESSION OF THE PIK3CA ONCOGENE LEADING TO INCREASED INVASION OFBASAL-LIKE BREAST CARCINOMA CELLS

Background: Basal-like breast carcinoma (BLBC) is the mostaggressive subtype of breast cancer. 73% of BLBC over-express YB-1, anoncogenic transcription/translation factor. PIK3CA, which codes for the p110? catalytic subunit ofphosphatidylinositol-3-kinase (PI3K), is another oncogene. The PI3K signalling pathway is fundamental in the regulation of many cellular functions and isoften deregulated in cancer. Despite its importance, the knowledge on the transcriptional regulation of PIK3CA is limited. Indeed, we have recently published the first report on the PIK3CA promoter. Methods and Results: A genome-wide chromatin immunoprecipitation on chip (ChIP-on-chip) analysis of a BLBC cell-line(SUM149) suggested binding of YB-1 to the PIK3CA promoter. This binding was verified using traditional chromatin immunoprecipitation (ChIP). Furthermore, electrophoretic mobility shift assay (EMSA) using oligonucleotides with eitherwild-type or mutated YB-1 responsive elements mapped YB-1 binding to three sites on the PIK3CA promoter. Silencing YB-1 in BLBC cell-lines (SUM149, HCC1937, andMDA-MB-231) decreased, while over-expression of YB-1 increased the PIK3CA promoter activity, transcript, and protein levels. Interestingly, array comparative genomic hybridization(aCGH) and quantitative PCR demonstrated PIK3CA copy number gains in HCC1937 andMDA-MB-231 cells. Although PIK3CA amplifications are overall uncommon (9%) in breast cancer, we demonstrated here that low level gains in PIK3CA copy number are present in 30%of primary BLBC cases. Furthermore, it has previously been demonstrated that mutations of PIK3CA are the most common genetic aberration (27%) found in breast cancer. These mutations lead to constitutive activation of p110? and are highly oncogenic. Over-expression of YB-1in MCF-7 cells, which harbour an activating PIK3CA mutation, increased PIK3CA transcript and protein levels. Furthermore, induction of PIK3CA by YB-1 leads to increased levels of urokinase plasminogenactivator (uPA) and invasion. Conclusions: Our data demonstrates that YB-1 binds to the PIK3CA promoter and induces itsexpression whether the gene is wild-type or amplified. Moreover, since YB-1induces expression of the active mutant p110?, then therapeutic inhibition of YB-1 may lead to decreased p110? and interference with the constitutively activated PI3K pathway in cancers. In addition, the YB-1/PIK3CA/uPA network provides information regarding the possible therapeutic targets for prevention of breast cancer invasion and metastasis. A.A. is supported by a Child and Family–CIHR–UBC MD/PhD Studentship Award

Multimodality imaging of the new generation bi- and tri-fusion vectors in living mice.

<p>1a1 and 1a2. Fluorescence and Bioluminescence imaging. 10×10⁶ of 293T cells transfected with <i>CMV-ttk (A), CMV-fluc2-tdt-ttk (B), CMV-fluc2(C),</i> and <i>CMV-tdt (D)</i> plasmids were implanted subcutaneously in living mice (n = 3) and were imaged first for fluorescence and then for bioluminescence after injection of D- luciferin using IVIS imaging system. Signals were seen only from the cells expressing <i>CMV-tdt</i> and <i>CMV-fluc2-tdt-ttk</i> vector (for fluorescence) and from cells expressing <i>CMV- fluc2</i> and <i>CMV-fluc2-tdt-ttk</i> reporter for bioluminescence. Signals were recorded as max (pixel/sec/cm²/steradian). 1a3. microPET imaging. Mice described in 1a1 were injected with 200 µCi of 18F-FHBG and microPET imaging was performed after 1 hr for 10 minutes. Specific uptake of 18F-FHBG was seen in cells expressing the <i>CMV-ttk</i> and <i>CMV-fluc2-tdt-ttk</i> vectors. High nonspecific accumulation of 18F-FHBG was seen in the gastro-intestinal tract (GI). 1a4. Coronal section of the same microPET image described in 1a3. 1b. Graphical representation of the quantified fluorescence (1b1), bioluminescence (1b2) and microPET (1b3) signals. 1b1 and 1b2. Fluorescence (B and D) and bioluminescence (B and C) signals were calculated for the respective ROIs drawn over the sites of implanted cells. The SEM represents 3 experiments (ns.- statistically non-significant and *indicates p<0.05). 1b3. Percent injected dose (%ID/G) of 18F-FHBG uptakes were calculated for the respective ROIs drawn over the implanted cell (A and B) which showed similar uptake (ns). 1c. Comparative analysis of the fluorescence activity of the new bi and trifusion vectors in cell culture: 293T cells were transiently transfected with <i>CMV-fluc2-tdt-ttk</i>, <i>CMV-mtfl-tdt-ttk, CMV-mtfl-tdt</i> and <i>CMV-fluc2-tdt</i> plasmids and FACS analysis was done from equal number of cells after 24 hrs. All the experiments were performed in triplicate (*indicates p<0.05). 1d & 1e. Fluorescence and bioluminescence imaging of the new bi and triple fusion vector. 10×10⁶ of 293T cells transfected with <i>CMV-fluc2-tdt-ttk</i>, <i>CMV-mtfl-tdt-ttk, CMV-mtfl-tdt</i> and <i>CMV-fluc2-tdt</i> plasmids were implanted on the dorsal side of a nude mouse and imaged for fluorescence (1d) using Maestro system and bioluminescence (1e) as described above. Cells expressing <i>CMV-fluc2-tdt</i> clearly exhibited highest fluorescence and bioluminescence signals among all group of cells.</p

In vitro studies on effect of Cisplatin and paclitaxel on PIK3CA promoter and p53.

<p>2a-d and 2k -2l. <i>PIK3CA</i> promoter activity in drug treated PA1-<i>PIK3CA-fluc2-tdt</i> (PPF), A2780-<i>PIK3CA-fluc2-tdt</i> (APFT) and SKOV3-<i>PIK3CA-fluc2-tdt</i> (SPFT) (2k and l) cells. A dose dependent decrease in luciferase activity (<i>PIK3CA</i> promoter activity) was observed on increasing concentrations of cisplatin treatments in PPF cells (at 3 & 5 µg/ml cisplatin, p<0.03) (2a) and in APFT cells at 20 and 30 µg/ml cisplatin (p<0.003) (2c). Similar dose dependent decrease in luciferase activity was obtained on increasing concentration of paclitaxel treatment in PPF up to 25 µg/ml, p<0.001) (2b) and in APFT cells upto 20 µg/ml paclitaxel, p<0.05 (2d) No such dose dependent change was observed on treatments with increasing concentration of cisplatin and paclitaxel (2k and 2l) in SPFT cells. 2e-2 h. Endogenous expression of p110α and p53 proteins after drug treatments. Western blot analysis of p53 protein from lysates of cisplatin and paclitaxel treated PPF and APFT cells showed induction in p53 level after 24hrs; however no change was observed after 2hrs. The p110α levels did not show any change after drug treatment (2e & 2g). The densitometric analysis representing the same is shown in 2d & 2h. The p53 mutant status in SPFT cells was verified by western blotting (2 m). 2i & 2j. Nuclear localization of p53 after treatment in cisplatin treated cells. p53 protein (red in 3g and green in 3h) showed nuclear localization upon treatment with cisplatin for 2hrs as compared to the vehicle treatment in PPF and APFT cells. DAPI (blue) indicated the nuclear staining.</p

The p53 binding sites in the PIK3CA promoter with core sequence (underlined) and the mutagenic primers.

<p>The p53 binding sites in the PIK3CA promoter with core sequence (underlined) and the mutagenic primers.</p

Transcription factor binding sites in PIK3CA promoter*.

<p>Note: *As analysed by the Genomatix software and using references from Astanehe et al, 2008 and Yang et al, 2009.</p

Non-invasive imaging of PIK3CA promoter modulation in tumor xenografts of living mice after cisplatin treatment.

<p>3a. Bioluminescence signal of PIK3CA promoter in PPF tumors. 3a1. Graphical representation of the kinetics of <i>PIK3CA</i> promoter modulation. The luciferase activity in the control group of mice (n = 3) increased with time while that of the treated group (n = 3) attenuated from the fourteenth day of treatment till the 22^nd day (end point) after one treatment (8 mg/kg) (shaded area represented the days of treatment). At 22^nd day, measurable attenuation in the bioluminescence signal between the control and treated mice was evident however it did not reach statistical significance. (Day 1 represents the day prior to treatment). 3a2. Graphical representation of fold-changes in the bioluminescence signals. The temporal fold change in bioluminescence signal (post treatment signal/pre treatment signal) demonstrated augmented bioluminescence in the control group and attenuation in the treated group. 3a3. Representative bioluminescent images of the mice bearing PPF tumors. Mouse from the control and treated group exhibited specific and similar intensity signals which decreased after treatment (arrowhead). 3b. Bioluminescence signal of PIK3CA promoter in APFT tumors after cisplatin treatment. 3b1. Graphical representation of the kinetics of <i>PIK3CA</i> promoter modulation. The bioluminescence signal in the control mice (n = 3) increased with time while that of the treated group (n = 4) showed a slight decrease at 11th day after first treatment and significant attenuation at 15^th day after second treatment (p<0.025). 3b2. Graphical representation of fold-changes in the bioluminescence signals. The temporal fold change in bioluminescence signals (post treatment signal/pre treatment signal) demonstrated augmented bioluminescence in the control group and but attenuation in the treated (Day 10 represented signal prior to treatment). 3b3. Representative bioluminescent images of the mice bearing APFT tumors. Mouse from control and treated group exhibited specific signals which decreased only in treated mouse as shown by an arrow.</p

Molecular decoy to the Y-box binding protein-1 suppresses the growth of breast and prostate cancer cells whilst sparing normal cell viability.

The Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor that is activated by phosphorylation at S102 whereby it induces the expression of growth promoting genes such as EGFR and HER-2. We recently illustrated by an in vitro kinase assay that a novel peptide to YB-1 was highly phosphorylated by the serine/threonine p90 S6 kinases RSK-1 and RSK-2, and to a lesser degree PKCα and AKT. Herein, we sought to develop this decoy cell permeable peptide (CPP) as a cancer therapeutic. This 9-mer was designed as an interference peptide that would prevent endogenous YB-1(S102) phosphorylation based on molecular docking. In cancer cells, the CPP blocked P-YB-1(S102) and down-regulated both HER-2 and EGFR transcript level and protein expression. Further, the CPP prevented YB-1 from binding to the EGFR promoter in a gel shift assay. Notably, the growth of breast (SUM149, MDA-MB-453, AU565) and prostate (PC3, LNCap) cancer cells was inhibited by ∼90% with the CPP. Further, treatment with this peptide enhanced sensitivity and overcame resistance to trastuzumab in cells expressing amplified HER-2. By contrast, the CPP had no inhibitory effect on the growth of normal immortalized breast epithelial (184htert) cells, primary breast epithelial cells, nor did it inhibit differentiation of hematopoietic progenitors. These data collectively suggest that the CPP is a novel approach to suppressing the growth of cancer cells while sparing normal cells and thereby establishes a proof-of-concept that blocking YB-1 activation is a new course of cancer therapeutics