Anna L. Stratford

Phosphorylated Insulin-Like Growth Factor-I/Insulin Receptor Is Present in All Breast Cancer Subtypes and Is Related to Poor Survival

Drugs that target the insulin-like growth factor-I receptor (IGF-IR) and/or insulin receptor (IR) are currently under investigation for a variety of malignancies including breast cancer. Although we have previously reported that IGF-IR expression in primary breast tumors is common, the activation status of this receptor has not been examined in relation to survival. Phosphorylated IGF-IR/IR (P-IGF-IR/IR) and its downstream signaling partner phospho-S6 (P-S6) were evaluated immunohistochemically in tumor tissue microarrays representing 438 cases of invasive breast cancer. P-IGF-IR/IR (n = 114; P = 0.046) and total levels of IR (n = 122; P = 0.009) were indicative of poor survival, whereas total IGF-IR (n = 112; P = 0.304) was not. P-IGF-IR/IR and P-S6 were coordinately expressed in primary breast tumors (likelihood ratio, 11.57; P = 6.70 x 10(-4)). Importantly, P-IGF-IR/IR was detected in all breast cancer subtypes (luminal, 48.1%; triple negative, 41.9%; and HER2, 64.3%). In vitro, the IGF-IR/IR inhibitor BMS-536924 decreased phospho-RSK and P-S6, and significantly suppressed the growth of breast cancer cell lines MCF-7, SUM149, and AU565 representing the luminal, triple negative, and HER2 subtypes, respectively, in monolayer and soft agar. BMS-536924 also inhibited growth in tamoxifen resistant MCF-7 Tam-R cells while having little effect on immortalized normal breast epithelial cells. Thus, we can determine which patients have the activated receptor and provide evidence that P-IGF-IR/IR is a prognostic factor for breast cancer. Beyond this, P-IGF-IR/IR could be a predictive marker for response to IGF-IR and/or IR-targeted therapies, as these inhibitors may be of benefit in all breast cancer subtypes including those with acquired resistance to tamoxifen.

Y-box binding protein-1 induces the expression of CD44 and CD49f leading to enhanced self-renewal, mammosphere growth, and drug resistance.

Y-box binding protein-1 (YB-1) is an oncogenic transcription/translation factor expressed in >40% of breast cancers, where it is associated with poor prognosis, disease recurrence, and drug resistance. We questioned whether this may be linked to the ability of YB-1 to induce the expression of genes linked to cancer stem cells such as CD44 and CD49f. Herein, we report that YB-1 binds the CD44 and CD49f promoters to transcriptionally upregulate their expressions. The introduction of wild-type (WT) YB-1 or activated P-YB-1(S102) stimulated the production of CD44 and CD49f in MDA-MB-231 and SUM 149 breast cancer cell lines. YB-1-transfected cells also bound to the CD44 ligand hyaluronan more than the control cells. Similarly, YB-1 was induced in immortalized breast epithelial cells and upregulated CD44. Conversely, silencing YB-1 decreased CD44 expression as well as reporter activity in SUM 149 cells. In mice, expression of YB-1 in the mammary gland induces CD44 and CD49f with associated hyperplasia. Further, activated mutant YB-1(S102D) enhances self-renewal, primary and secondary mammosphere growth, and soft-agar colony growth, which were reversible via loss of CD44 or CD49f. We next addressed the consequence of this system on therapeutic responsiveness. Here, we show that paclitaxel induces P-YB-1(S102) expression, nuclear localization of activated YB-1, and CD44 expression. The overexpression of WT YB-1 promotes mammosphere growth in the presence of paclitaxel. Importantly, targeting YB-1 sensitized the CD44(High)/CD24(Low) cells to paclitaxel. In conclusion, YB-1 promotes cancer cell growth and drug resistance through its induction of CD44 and CD49f.

Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a potential target for therapy

IntroductionBasal-like breast cancers (BLBCs) are very aggressive, and present serious clinical challenges as there are currently no targeted therapies available. We determined the regulatory role of Y-box binding protein-1 (YB-1) on epidermal growth factor receptor (EGFR) overexpression in BLBC, and the therapeutic potential of inhibiting EGFR. We pursued this in light of our recent work showing that YB-1 induces the expression of EGFR, a new BLBC marker.MethodsPrimary tumour tissues were evaluated for YB1 protein expression by immunostaining tissue microarrays, while copy number changes were assessed by comparative genomic hybridization (CGH). The ability of YB-1 to regulate EGFR was evaluated using luciferase reporter, chromatin immunoprecipitation (ChIP) and gel shift assays. The impact of Iressa on monolayer cell growth was measured using an ArrayScan VTI high-throughput analyser to count cell number, and colony formation in soft agar was used to measure anchorage-independent growth.ResultsYB-1 (27/37 or 73% of cases, P = 3.899 × 10-4) and EGFR (20/37 or 57.1% of cases, P = 9.206 × 10-12) are expressed in most cases of BLBC. However, they are not typically amplified in primary BLBC, suggesting overexpression owing to transcriptional activation. In support of this, we demonstrate that YB-1 promotes EGFR reporter activity. YB-1 specifically binds the EGFR promoter at two different YB-1-responsive elements (YREs) located at -940 and -968 using ChIP and gel shift assays in a manner that is dependent on the phosphorylation of S102 on YB-1. Inhibiting EGFR with Iressa suppressed the growth of SUM149 cells by ~40% in monolayer, independent of mutations in the receptor. More importantly anchorage-independent growth of BLBC cell lines was inhibited with combinations of Iressa and YB-1 suppression.ConclusionWe have identified for the first time a causal link for the expression of EGFR in BLBC through the induction by YB-1 where it binds specifically to two distinguished YREs. Finally, inhibition of EGFR in combination with suppression of YB-1 presents a potential opportunity for therapy in BLBC.

Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells

IntroductionBasal-like breast cancers (BLBC) frequently overexpress the epidermal growth factor receptor (EGFR) and subsequently have high levels of signaling through the MAP kinase pathway, which is thought to contribute to their aggressive behavior. While we have previously reported the expression of Y-box binding protein-1 (YB-1) in 73% of BLBC, it is unclear whether it can be regulated by a component of the MAP kinase signaling pathway. Phosphorylation of YB-1 at the serine 102 residue is required for transcriptional activation of growth-enhancing genes, such as EGFR. Using Motifscan we identified p90 ribosomal S6 kinase (RSK) as a potential candidate for activating YB-1.MethodsInhibition of RSK1 and RSK2 was achieved using siRNA and the small molecule SL0101. RSK1, RSK2, activated RSK and kinase-dead RSK were expressed in HCC1937 cells. Kinase assays were performed to illustrate direct phosphorylation of YB-1 by RSK. The impact of inhibiting RSK on YB-1 function was measured by luciferase assays and chromatin immunoprecipitation.ResultsUsing an in vitro kinase assay, RSK1 and RSK2 were shown to directly phosphorylate YB-1. Interestingly, they were more effective activators of YB-1 than AKT or another novel YB-1 kinase, PKCα. Phosphorylation of YB-1 (serine 102 residue) is blocked by inhibition of the MAP kinase pathway or by perturbing RSK1/RSK2 with siRNA or SL0101. In immortalized breast epithelial cells where RSK is active yet AKT is not, YB-1 is phosphorylated. Supporting this observation, RSK2-/- mouse embryo fibroblasts lose the ability to phosphorylate YB-1 in response to epidermal growth factor. This subsequently interfered with the ability of YB-1 to regulate the expression of EGFR. The RSK inhibitor SL0101 decreased the ability of YB-1 to bind the promoter, transactivate and ultimately reduce EGFR expression. In concordance with these results the expression of constitutively active RSK1 increased YB-1 phosphorylation, yet the kinase-dead RSK did not.ConclusionsWe therefore conclude that RSK1/RSK2 are novel activators of YB-1, able to phosphorylate the serine 102 residue. This provides a newly described mechanism whereby YB-1 is activated in breast cancer. This implicates the EGFR/RSK/YB-1 pathway as an important component of BLBC, providing an important opportunity for therapeutic intervention.

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.

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

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

PTTG and PBF repress the human sodium iodide symporter.

The ability of the thyroid to accumulate iodide provides the basis for radioiodine ablation of differentiated thyroid cancers and their metastases. Most thyroid tumours exhibit reduced iodide uptake, although the mechanisms accounting for this remain poorly understood. Pituitary tumour transforming gene (PTTG) is a proto-oncogene implicated in the pathogenesis of thyroid tumours. We now show that PTTG and its binding factor PBF repress expression of sodium iodide symporter (NIS) messenger RNA (mRNA), and inhibit iodide uptake. This process is mediated at least in part through fibroblast growth factor-2. In detailed studies of the NIS promoter in rat FRTL-5 cells, PTTG and PBF demonstrated specific inhibition of promoter activity via the human upstream enhancer element (hNUE). Within this ∼1 kb element, a complex PAX8-upstream stimulating factor 1 (USF1) response element proved critical both to basal promoter activity and to PTTG and PBF repression of NIS. In particular, repression by PTTG was contingent upon the USF1, but not the PAX8, site. Finally, in human primary thyroid cells, PTTG and PBF similarly repressed the NIS promoter via hNUE. Taken together, our data suggest that the reported overexpression of PTTG and PBF in differentiated thyroid cancer has profound implications for activity of the NIS gene, and hence significantly impacts upon the efficacy of radioiodine treatment.

Targeting p90 Ribosomal S6 Kinase Eliminates Tumor-Initiating Cells by Inactivating Y-Box Binding Protein-1 in Triple-Negative Breast Cancers†‡§

Y‐box binding protein‐1 (YB‐1) is the first reported oncogenic transcription factor to induce the tumor‐initiating cell (TIC) surface marker CD44 in triple‐negative breast cancer (TNBC) cells. In order for CD44 to be induced, YB‐1 must be phosphorylated at S102 by p90 ribosomal S6 kinase (RSK). We therefore questioned whether RSK might be a tractable molecular target to eliminate TICs. In support of this idea, injection of MDA‐MB‐231 cells expressing Flag‐YB‐1 into mice increased tumor growth as well as enhanced CD44 expression. Despite enrichment for TICs, these cells were sensitive to RSK inhibition when treated ex vivo with BI‐D1870. Targeting RSK2 with small interfering RNA (siRNA) or small molecule RSK kinase inhibitors (SL0101 and BI‐D1870) blocked TNBC monolayer cell growth by ∼100%. In a diverse panel of breast tumor cell line models RSK2 siRNA predominantly targeted models of TNBC. RSK2 inhibition decreased CD44 promoter activity, CD44 mRNA, protein expression, and mammosphere formation. CD44+ cells had higher P‐RSKS221/227, P‐YB‐1S102, and mitotic activity relative to CD44− cells. Importantly, RSK2 inhibition specifically suppressed the growth of TICs and triggered cell death. Moreover, silencing RSK2 delayed tumor initiation in mice. In patients, RSK2 mRNA was associated with poor disease‐free survival in a cohort of 244 women with breast cancer that had not received adjuvant treatment, and its expression was highest in the basal‐like breast cancer subtype. Taking this further, we report that P‐RSKS221/227 is present in primary TNBCs and correlates with P‐YB‐1S102 as well as CD44. In conclusion, RSK2 inhibition provides a novel therapeutic avenue for TNBC and holds the promise of eliminating TICs. STEM CELLS2012;30:1338–1348

YB-1 evokes susceptibility to cancer through cytokinesis failure, mitotic dysfunction, and HER2 amplification

Y-box binding protein-1 (YB-1) expression in the mammary gland promotes breast carcinoma that demonstrates a high degree of genomic instability. In the present study, we developed a model of pre-malignancy to characterize the role of this gene during breast cancer initiation and early progression. Antibody microarray technology was used to ascertain global changes in signal transduction following the conditional expression of YB-1 in human mammary epithelial cells (HMEC). Cell cycle-associated proteins were frequently altered with the most dramatic being LIM kinase 1/2 (LIMK1/2). Consequently, the misexpression of LIMK1/2 was associated with cytokinesis failure that acted as a precursor to centrosome amplification. Detailed investigation revealed that YB-1 localized to the centrosome in a phosphorylation-dependent manner, where it complexed with pericentrin and γ-tubulin. This was found to be essential in maintaining the structural integrity and microtubule nucleation capacity of the organelle. Prolonged exposure to YB-1 led to rampant acceleration toward tumorigenesis, with the majority of cells acquiring numerical and structural chromosomal abnormalities. Slippage through the G1/S checkpoint due to overexpression of cyclin E promoted continued proliferation of these genomically compromised cells. As malignancy further progressed, we identified a subset of cells harboring HER2 amplification. Our results recognize YB-1 as a cancer susceptibility gene, with the capacity to prime cells for tumorigenesis.

YB-1 is a Transcription/Translation Factor that Orchestrates the Oncogenome by Hardwiring Signal Transduction to Gene Expression

Conventional gene expression profiling relies on using fluorescent detection of hybridized probes. Physical characteristics of fluorophores impose limitations on achieving a highly multiplex gene analysis of single cells. Our work demonstrates the feasibility of using metal-tagged in situ hybridization for mRNA detection by inductively coupled plasma mass spectrometry (ICP-MS). ICP-MS as an analytical detector has a number of unique and relevant properties: 1) metals and their stable isotopes generate non-overlapping distinct signals that can be detected simultaneously; 2) these signals can be measured over a wide dynamic range; 3) ICP-MS is quantitative and very sensitive. We used commercial antibodies conjugated to europium (Eu) and gold together with biotinylated oligonucleotide probes reacted with terbium-labeled streptavidin to demonstrate simultaneous mRNA and protein detection by ICP-MS in leukemia cells.Urokinase-type plasminogen activator (uPA) is associated with cancer recurrence where the most evidence comes from studies in breast cancer. According to the European Organization for Research and Treatment of Cancer, uPA is considered one of the most prominent biomarkers for cancer recurrence and therefore new agents are needed to inhibit it. Whether uPA is also expressed in pediatric cancers is yet unknown. If it is then uPA inhibitors might also help children with recurrent cancers. In this study, we addressed whether the integrin-linked kinase inhibitor (ILK), QLT0267, could suppress uPA. We previously showed that uPA expression is maximally inhibited when both the Akt and MAP kinase pathways were blocked which we anticipated can be achieved via QLT0267. In MDA-MB-231 breast cancer cells, QLT0267 blocked signaling through Akt and MAP kinase with a correlative decrease in uPA protein and mRNA, which corresponded to an inhibition of c-Jun phosphorylation. Consistent with these findings, cellular invasion was inhibited with either QLT0267 or with small interfering RNA against ILK. We then questioned whether uPA was commonly expressed in childhood sarcomas and if QLT0267 might be effective in this setting. We determined for the first time that uPA was highly expressed in rhabdomyosarcomas (RMS), but not Ewings sarcomas by screening cell lines (n = 31) and patient samples (n = 200) using Affymetrix microarrays. In alveolar RMS (ARMS) cell lines, QLT0267 blocked cell signaling, uPA production, invasion and ultimately survival. We concluded that QLT0267 blocks the production of uPA providing a new target for the management of recurrent cancers.There is a growing body of literature suggesting that signaling based therapy might be a potential approach for medullary thyroid cancer (MTC). In this review we focus on the tumor suppressor role of Notch1 and Raf-1 signaling in MTC. Interestingly these two pathways are minimally active or absent in these tumors and activation of Notch1 and Raf-1 significantly reduces tumor growth in vitro. Therefore, identification of compounds that induce these pathways could be a potential strategy to treat patients with MTC.In the last two decades there has been considerable progress in our understanding of the role of sphingolipids in controlling signal transduction processes, particularly in the mechanisms leading to regulation of cell growth and death. Ceramide is a well-characterized sphingolipid metabolite and second messenger that can be produced by cancer cells in response to a variety of stimuli, including therapeutic drugs, leading to cell cycle arrest and apoptosis. Although this is a promising aspect when thinking of treating cancer, it should be borne in mind that ceramide production may not always be a growth inhibitory or pro-apoptotic signal. In fact, ceramide can be readily converted to sphingosine 1-phosphate (S1P) by the concerted actions of ceramidases and sphingosine kinases, or to ceramide 1-phosphate (C1P) by the action of ceramide kinase. In general, S1P and C1P have opposing effects to ceramide, acting as pro-survival or mitogenic signals in most cell types. This review will address our current understanding of the many roles of ceramide, S1P and C1P in the regulation of cell growth and survival with special emphasis to the emerging role of these molecules and their metabolizing enzymes in controlling tumor progression and metastasis.The Y-box Binding Protein-1 (YB-1) is a highly conserved oncogenic transcription/translation factor that is expressed in cancers affecting adults and children. It is now believed that YB-1 plays a causal role in the development of cancer given recent work showing that its expression drives the tumorigenesis in the mammary gland. In human breast cancers, YB-1 is associated with rapidly proliferating tumors that are highly aggressive. Moreover, expression of YB-1 promotes the growth of breast cancer cell lines both in monolayer and anchorage independent conditions. The involvement of YB-1 in breast cancer pathogenesis has made it a putative therapeutic target; however, the mechanism(s) that regulate YB-1 are poorly understood. This review first describes the oncogenic properties of YB-1 in cancer. It also highlights the importance of YB-1 in hardwiring signal transduction pathways to the regulation of genes involved in the development of cancer.Specific combinations of transcription-factor binding sites in the promoter regions of genes regulate gene expression, and thus key functional processes in cells. Analysis of such promoter regions in specific functional contexts can be used to delineate novel disease-associated genes based on shared phenotypic properties. The aim of this study was to utilize promoter analysis to predict cell proliferation-associated genes and to test this method in colon cancer cell lines. We used freely-available bioinformatic techniques to identify cell-proliferation-associated genes expressed in colon cancer, extract a shared promoter module, and identify novel genes that also contain this module in the human genome. An EGRF/ETSF promoter module was identified as prevalent in proliferation-associated genes from a colon cancer cDNA library. We detected 30 other genes, from the known promoters of the human genome, which contained this proliferation-associated module. This group included known proliferation-associated genes, such as HERG1 and MCM7, and a number of genes not previously implicated in cell proliferation in cancer, such as TSPAN3, Necdin and APLP2. Suppression of TSPAN3 and APLP2 by siRNA was performed and confirmed by RT-PCR. Inhibition of these genes significantly inhibited cell proliferation in colon cancer cell lines. This study demonstrates that promoter analysis can be used to identify novel cancer-associated genes based on shared functional processes.Background: Potassium bromate (KBrO3), used in both the food and cosmetics industry, and a drinking water disinfection by-product, is a nephrotoxic compound and rodent carcinogen. To gain insight into the carcinogenic mechanism of action and provide possible biomarkers of KBrO3 exposure, the gene expression in kidneys from chronically exposed male F344 rats was investigated. Methods: Male F344 rats were exposed to KBrO3 in drinking water for 52 and 100 wk. Kidneys were removed, frozen, and stored at −80°C, then used for Affymetrix microarray analysis. Gene expression patterns were examined using a non-carcinogenic (20 ppm) and carcinogenic dose (400 ppm) at 52 wk, and compared to 100 wk high dose (400 ppm) and adenoma gene expression. Results: Statistical analysis revealed 144, 224, 43, and 994 genes out of 15866 from the 52 wk low, 52 wk high, 100 wk high, and adenomas respectively, were differentially expressed when compared to control kidneys. Gene ontology classification of the 52 wk high dose showed alterations of gene transcripts involved in oxidative stress, lipid metabolism, kidney function/ion transport, and cellular function. In a comparison of kidney development gene expression, alterations were seen in the adenomas but not in the 52 wk bromate-treated kidneys. However, the normal kidney from the high dose group resembled the adenoma expression pattern with early kidney development genes being up-regulated and adult phase genes being down-regulated. Moreover, eight genes were identified which could serve as biomarkers of carcinogenic exposure to bromate. The most promising of these was Pendrin, or Slc26a4, a solute carrier of chloride and iodide active in the kidney, thyroid, and inner ear. All these tissues are targets of KBrO3 toxicity. Expression array results were verified with quantitative real-time rtPCR. Conclusions: These data demonstrate that the 400 ppm carcinogenic dose of KBrO3 showed marked gene expression differences from the 20 ppm non-carcinogenic dose. Comparison of kidney development gene expression showed that the adenoma patterns were more characteristic of embryonic than adult kidneys, and that the normal kidney from the high dose group resembled the adenoma-like gene expression pattern. Taken together, the analysis from this study identifies potential biomarkers of exposure and illuminates a possible carcinogenic mode of action for KBrO3.Chronic eosinophilic leukemia is a clonal disease characterized by hypereosinophilia and eosinophilia-related pathologic manifestations. Recently, the fusion gene FIP1L1/PDGFRA was found in the long arm of chromosome 4 and its expression has been shown to be associated with development of a clinical hypereosinophilic syndrome (HES) in a significant proportion of patients. FIP1L1/PDGFRα, the product of the gene FIP1L1/PDGFRA, is a constitutively activated tyrosine kinase and can be inhibited by imatinib mesylate. Several investigations have tried to dissect the mechanism of leukemogenesis and signaling induced by FIP1L1/PDGFRα in cell lines, primary human eosinophils and in murine myeloproliferative models. In this review, we analyzed the current knowledge on the relationship between FIP1L1/PDGFRα-induced signaling and eosinophil proliferation, survival and activation, specially focusing on its possible role in the modulation of cytokine and chemoattractant signaling pathways.The increasing number of proteomic and DNA-microarray studies is continually providing a steady acquisition of data on the molecular abnormalities associated with human tumors. Rapid translation of this accumulating biological information into better diagnostics and more effective cancer therapeutics in the clinic depends on the use of robust function-testing strategies. Such strategies should allow identification of molecular lesions that are essential for the maintenance of the transformed phenotype and enable validation of potential drug-targets. The tetracycline regulated gene expression/ suppression systems (Tet-systems) developed and optimized by bioengineers over recent years seem to be very well suited for the function-testing purposes in cancer research. We review the history and latest improvements in Tet-technology in the context of functional oncogenomics.Multiple lines of evidence implicate over-expression and activation of the androgen receptor (AR) in the progression of prostate cancer (PC) to androgen-independence (AI) and resistance to therapy. The mechanisms leading to AR over-expression are not fully understood but binding of Sp1 to specific Sp1-binding sites in the AR promoter and 5′-untranslated region (5′-UTR) was shown to up-regulate AR transcription. In this work, we further characterized the role of Sp1 in the control of AR transcription and explored its potential as a therapeutic target in androgen-dependent (AD) and independent (AI) LNCaP cells. We identified a pair of new Sp1-binding site in the 5′-UTR of AR which we named ARSp1-3. ARSp1-3 binds Sp1 with higher affinity than other known Sp1-binding sites in the promoter/5′-UTR and in transfection experiments, the ARSp1-3 reporter showed higher transcriptional activity in AI than in AD cells. Treatment of these cells with nanomolar concentrations of Mithramycin inhibited binding of Sp1 to its binding sites in the promoter/5′-UTR of the AR gene but more specifically the binding of ARSp1-3 while other regulatory elements of the AR promoter were not affected. Inhibition of Sp1 binding by Mithramycin decreased the AR transcription and transactivation of PSA reporter constructs. At the lowest concentrations, Mithramycin decreased endogenous AR protein and proliferation of AD and AI LNCaP cells. The combinations of Mithramycin with either paclitaxel or bicalutamide were highly synergistic. Conclusion: Sp1 binding induces AR transcription in LNCaP cells. The higher affinity of ARSp1-3 for Sp1 may support higher AR mRNA levels in AI than AD LNCaP cells. Mithramycin is a potent and specific inhibitor of Sp1 and AR transcription with potential, at very low concentrations, to enhance the efficacy of hormones or taxane based therapy in patients with recurrent or androgen-independent progression that sustain AR expression.Many tumor markers for bladder cancer have been evaluated for use in detecting and monitoring bladder cancers tissue specimens, bladder washes, and urine specimens. However, none of the biomarkers reported to date has shown sufficient sensitivity and specificity to detect the entire spectrum of bladder cancers in routine clinical practice. The limited value of the established prognostic markers demands analysis of new molecular parameters having the potential to predict the prognosis of bladder cancer patients, particularly, the high-risk patients at risk of cancer progression and recurrence. Abnormal methylation of CpG islands can efficiently repress transcription of the associated gene in a manner akin to mutations and deletions. Several tumor suppressor genes correlated with bladder cancer contain CpG islands in their promoters. Markers for aberrant methylation may be a potential gateway for monitoring bladder cancer. Hypermethylation of several gene promoters was detected in urine sediment DNA from bladder cancer patients. Detection of DNA methylation in voided urine is feasible and noninvasive. Methylation is an important molecular mechanism in the development of bladder cancer and could be used as a prognostic and diagnostic marker. Aberrant patterns of epigenetic modification could, in the near future, be crucial indicators in cancer diagnosis, prognosis, and may additionally be good targets for developing novel therapies while maintaining quality of life.In 1911 Peyton Rous described a transmissible agent that could induce sarcoma in chicken, this was later identifi ed as a virus and named Rous Sarcoma Virus (Rous, 1911). Identifi cation of the viral tyrosine kinase v-Src and its cellular counterpart c-Src (later in the text referred as Src), introduced the concept of proto-oncogene which has had a signifi cant impact on the progress of our knowledge of carcinogenesis (Martin, 2001). Since its description, Src has been implicated in a variety of malignancies (Frame, 2002) including prostate cancer (Chang et al. 2007), which is the most commonly diagnosed cancer in men and the second leading cause of cancer-related death in men in the U.K. and U.S. (Jemal et al. 2007). The Src-family kinases (SFK) comprises of nine members including Src, Fyn, Yes, Blk, Yrk, Fgr, Hck, Lck and Lyn; Src, Fyn and Yes being ubiquitously expressed in all cells while other kinases are tissue specifi c. Apart from Src, two other family members, Fgr (Edwards et al. 2003) and Lyn (Goldenberg-Furmanov et al. 2004) have been implicated in prostate cancer. All SFK members share similar structure; each protein consists of four Src homology (SH) domains and a unique amino-terminal domain. High resolution crystallographic analysis of Src revealed the complex nature of structural changes involved in switching between active and inactive state. Src can be locked in an inactive conformation when its negative regulatory tail is phosphorylated at tyrosine Y530 by c-terminal Src kinase (Csk). However, when Src becomes autophosphorylated at tyrosine Y419, which is located in the kinase domain, the protein unfolds assuming its catalytically active conformation. Apart from being a tyrosine kinase, Src may function as a scaffolding molecule being an adaptor for other intracellular proteins that in turn can activate Src by the release of its intramolecular bonds. Another mechanism of Src activation, called peripheral targeting, involves translocation of inactive Src, which is located in the perinuclear region, to the cell periphery where Src becomes attached to the inner surface of cell membrane by its myristoylation fragment (Frame, 2002).Src interacts with a wide variety of proteins including receptor tyrosine kinases, G-protein coupled receptors, steroid receptors, integrins, other non-receptor protein kinases etc., which is re fl ected in the multiplicity of resulting cellular biological events (Thomas and Brugge, 1997). Crosstalk between Src and the components of PI3K (phosphatidylinositol 3-kinase) and MAPK (mitogen activated protein kinase) pathways may affect tumor cell proliferation and apoptosis while involvement in focal adhesion complexes, especially FAK (focal adhesion kinase), paxillin and p130CAS (p130 Crk-associate sub-strate) plays an important part in promoting cell adhesion, migration and invasion (Summy and Gallick, 2006). Considering its unique position at the crossroads of the intracellular signaling networks, Src has become an attractive target in the search of novel prostate cancer therapies (McCarty, 2004).The oncogene MCTS1, discovered as an amplified product in a subset of T-cell lymphoma lines, has been implicated in cell cycle progression and conferring a growth advantage in lymphomas and breast cancer. Recent research shows that it modulates the MAPK pathway and acts as a translational activator both in vivo and in vitro. In breast cancer cells, expression of MCTS1 confers aggressive properties and inhibits apoptosis. This article will review these data and its implications on our understanding of cancer.