Targeted Polo-like Kinase Inhibition Combined With Aurora Kinase Inhibition in Pediatric Acute Leukemia Cells.

BACKGROUND: Recent studies have shown that cell cycle events are tightly controlled by complex and shared activities of a select group of kinases. Among these, polo-like kinases (Plks) are regulatory mitotic proteins that are overexpressed in several types of cancer and are associated with poor prognosis. MATERIALS AND METHODS: We have evaluated, in preclinical in vitro studies, the activity of a panel of Plk inhibitors against cell lines derived from refractory pediatric leukemia, as well as primary leukemia cells, in culture. Through in vitro growth inhibition studies, Western blot analysis for the expression and activation of key regulators of cell growth and survival and gene silencing studies, we specifically examined the ability of these agents to induce cytotoxicity through the activation of apoptosis and their capacity to interact and modulate the expression and phosphorylation of Aurora kinases. RESULTS: Our findings show that the various Plk-1 inhibitors in development show potential utility for the treatment of pediatric leukemia and exhibit a wide range of phosphorylation and target modulatory capabilities. Finally, we provide evidence for a complex interregulatory relationship between Plk-1 and Aurora kinases enabling the identification of synergy and biologic correlates of drug combinations targeting the 2 distinct enzyme systems. DISCUSSION: This information provide the rationale for the evaluation of Plk-1 as an effective target for therapeutics in refractory pediatric leukemia and indicate compensatory activities between Plk-1 and Aurora kinases, providing insight into some of the complex mechanisms involved in the process of cell division.

Concise review: bullseye: targeting cancer stem cells to improve the treatment of gliomas by repurposing disulfiram.

Cancer stem cells (CSCs) are thought to be at the root of cancer recurrence because they resist conventional therapies and subsequently reinitiate tumor cell growth. Thus, targeting CSCs could be the bullseye to successful cancer therapeutics in the future. Brain tumors are some of the most challenging types of cancer to treat and the median survival following the initial diagnosis is 12-18 months. Among the different types of brain tumors, glioblastoma (GBM) is considered the most aggressive and remains extremely difficult to treat. Despite surgery, radiation, and chemotherapy, most patients develop refractory disease. Temozolomide (TMZ) is a chemotherapy used to treat GBM however resistance develops in most patients. The underlying mechanisms for TMZ resistance (TMZ-resistant) involve the expression of DNA repair gene O(6)-methylguanine-DNA methyltransferase. CSC genes such as Sox-2, BMI-1, and more recently Y-box binding protein-1 also play a role in resistance. In order to develop novel therapies for GBM, libraries of small interfering RNAs and off-patent drugs have been screened. Over the past few years, several independent laboratories identified disulfiram (DSF) as an off-patent drug that kills GBM CSCs. Reportedly DSF has several modes of action including its ability to inhibit aldehyde dehydrogenases, E3 ligase, polo-like kinase 1, and NFkB. Due to the fact that GBM is a disease of heterogeneity, chemotherapy with multitargeting properties may be the way of the future. In broader terms, DSF kills CSCs from a range of different cancer types further supporting the idea of repurposing it for "target practice."

EZH2 expression is a prognostic factor in childhood intracranial ependymoma: a Canadian Pediatric Brain Tumor Consortium study.

BACKGROUND: The cure rate for childhood intracranial ependymoma is approximately 70% in the setting of a gross total resection followed by radiation, but management remains challenging in patients with residual disease. Therefore, robust biomarkers are needed to guide the development of new targeted therapy. The authors evaluated the expression of several biomarkers in pediatric intracranial ependymoma and observed that the expression of enhancer of zeste homolog 2 (EZH2), a polycomb complex protein involved in epigenetic regulation of gene expression, was independently associated with poor survival. METHODS: Tissue microarray immunostaining was performed on 180 ependymoma samples from 12 of 16 Canadian pediatric centers. Expression levels of EZH2, Ki-67, B lymphoma Moloney-murine leukemia virus insertion region 1 homolog, tumor protein 16 (P16), Y-box binding protein 1, phosphorylated protein kinase B (pAKT), and epidermal growth factor receptor were evaluated. Cox regression analyses were performed, and the Kaplan-Meier method was used to construct survival curves. RESULTS: EZH2 expressed in 16% of tumors was associated with inferior 5-year overall survival. Ki-67 and pAKT levels were associated with a poor outcome in patients with posterior fossa ependymoma, and the absence of P16 was associated with a poor outcome in patients with supratentorial ependymoma. Multivariate analysis revealed that younger age and EZH2 expression (95% confidence interval, 1.1-36.0) were independent markers of a poor prognosis. CONCLUSIONS: EZH2 is a novel, independent marker of a poor prognosis in patients with ependymoma, especially in those who have tumors located in the posterior fossa. EZH2, pAKT, and P16 are potential therapeutic targets, particularly for patients who have tumors in which standard gross total resection plus fractionated radiotherapy is not feasible.

YB-1 transforms human mammary epithelial cells through chromatin remodeling leading to the development of basal-like breast cancer.

There is growing evidence that cancer-initiation could result from epigenetic changes. Y-box binding protein-1 (YB-1) is a transcription/translation factor that promotes the formation of tumors in transgenic mice; however, the underlying molecular events are not understood. To explore this in a human model system, YB-1 was expressed in mammary epithelial cells under the control of a tetracycline-inducible promoter. The induction of YB-1 promoted phenotypes associated with malignancy in three-dimensional breast acini cultures. This was attributed to YB-1 enhancing the expression and activity of the histone acetyltransferase p300 leading to chromatin remodeling. Specifically, this relaxation of chromatin allowed YB-1 to bind to the BMI1 promoter. The induction of BMI1 engaged the Polycomb complex resulting in histone H2A ubiquitylation and repression of the CDKN2A locus. These events manifested functionally as enhanced self-renewal capacity that occurred in a BMI1-dependent manner. Conversely, p300 inhibition with anacardic acid prevented YB-1 from binding to the BMI1 promoter and thereby subverted self-renewal. Despite these early changes, full malignant transformation was not achieved until RSK2 became overexpressed concomitant with elevated human telomerase reverse transcriptase (hTERT) activity. The YB-1/RSK2/hTERT expressing cells formed tumors in mice that were molecularly subtyped as basal-like breast cancer. We conclude that YB-1 cooperates with p300 to allow BMI1 to over-ride p16(INK4a) -mediated cell cycle arrest enabling self-renewal and the development of aggressive breast tumors.

FoxG1 interacts with Bmi1 to regulate self-renewal and tumorigenicity of medulloblastoma stem cells.

Brain tumors represent the leading cause of childhood cancer mortality, of which medulloblastoma (MB) is the most frequent malignant tumor. Recent studies have demonstrated the presence of several MB molecular subgroups, each distinct in terms of prognosis and predicted therapeutic response. Groups 1 and 2 are characterized by relatively good clinical outcomes and activation of the Wnt and Shh pathways, respectively. In contrast, groups 3 and 4 ("non-Shh/Wnt MBs") are distinguished by metastatic disease, poor patient outcome, and lack a molecular pathway phenotype. Current gene expression platforms have not detected brain tumor-initiating cell (BTIC) self-renewal genes in groups 3 and 4 MBs as BTICs typically comprise a minority of tumor cells and may therefore go undetected on bulk tumor analyses. Since increasing BTIC frequency has been associated with increasing tumor aggressiveness and poor patient outcome, we investigated the subgroup-specific gene expression profile of candidate stem cell genes within 251 primary human MBs from four nonoverlapping MB transcriptional databases (Amsterdam, Memphis, Toronto, Boston) and 74 NanoString-subgrouped MBs (Vancouver). We assessed the functional relevance of two genes, FoxG1 and Bmi1, which were significantly enriched in non-Shh/Wnt MBs and showed these genes to mediate MB stem cell self-renewal and tumor initiation in mice. We also identified their transcriptional regulation through reciprocal promoter occupancy in CD15+ MB stem cells. Our work demonstrates the application of stem cell data gathered from genomic platforms to guide functional BTIC assays, which may then be used to develop novel BTIC self-renewal mechanisms amenable to therapeutic targeting.

Overcoming resistance to Sonic Hedgehog inhibition by targeting p90 ribosomal S6 kinase in pediatric medulloblastoma.

BACKGROUND: Molecular subtyping has allowed for the beginning of personalized treatment in children suffering from medulloblastoma (MB). However, resistance inevitably emerges against these therapies, particularly in the Sonic Hedgehog (SHH) subtype. We found that children with SHH subtype have the worst outcome underscoring the need to identify new therapeutic targets. PROCEDURE: High content screening of a 129 compound library identified agents that inhibited SHH MB growth. Lead molecular target levels, p90 ribosomal S6 kinase (RSK) were characterized by immunoblotting and qRT-PCR. Comparisons were made to human neural stem cells (hNSC). Impact of inhibiting RSK with the small molecule BI-D1870 or siRNA was assessed in growth assays (monolayer, neurosphere, and soft agar). NanoString was used to detect RSK in a cohort of 66 patients with MB. To determine BI-D1870 pharmacokinetics/pharmacodynamics, 100 mg/kg was I.P. injected into mice and tissues were collected at various time points. RESULTS: Daoy, ONS76, UW228, and UW426 MB cells were exquisitely sensitive to BI-D1870 but unresponsive to SHH inhibitors. Anti-tumor growth corresponded with inactivation of RSK in MB cells. BI-D1870 had no effect on hNSCs. Inhibiting RSK with siRNA or BI-D1870 suppressed growth, induced apoptosis, and sensitized cells to SHH agents. Notably, RSK expression is correlated with SHH patients. In mice, BI-D1870 was well-tolerated and crossed the blood-brain barrier (BBB). CONCLUSIONS: RSK inhibitors are promising because they target RSK which is correlated with SHH patients as well as cause high levels of apoptosis to only MB cells. Importantly, BI-D1870 crosses the BBB, acting as a scaffold for development of more long-lived RSK inhibitors.

YB-1: oncoprotein, prognostic marker and therapeutic target?

Hanahan and Weinberg have proposed the 'hallmarks of cancer' to cover the biological changes required for the development and persistence of tumours [Hanahan and Weinberg (2011) Cell 144, 646-674]. We have noted that many of these cancer hallmarks are facilitated by the multifunctional protein YB-1 (Y-box-binding protein 1). In the present review we evaluate the literature and show how YB-1 modulates/regulates cellular signalling pathways within each of these hallmarks. For example, we describe how YB-1 regulates multiple proliferation pathways, overrides cell-cycle check points, promotes replicative immortality and genomic instability, may regulate angiogenesis, has a role in invasion and metastasis, and promotes inflammation. We also argue that there is strong and sufficient evidence to suggest that YB-1 is an excellent molecular marker of cancer progression that could be used in the clinic, and that YB-1 could be a useful target for cancer therapy.

Polo-like kinase 1 inhibition kills glioblastoma multiforme brain tumor cells in part through loss of SOX2 and delays tumor progression in mice.

Glioblastoma multiforme (GBM) ranks among the deadliest types of cancer and given these new therapies are urgently needed. To identify molecular targets, we queried a microarray profiling 467 human GBMs and discovered that polo-like kinase 1 (PLK1) was highly expressed in these tumors and that it clustered with the proliferative subtype. Patients with PLK1-high tumors were more likely to die from their disease suggesting that current therapies are inactive against such tumors. This prompted us to examine its expression in brain tumor initiating cells (BTICs) given their association with treatment failure. BTICs isolated from patients expressed 110-470 times more PLK1 than normal human astrocytes. Moreover, BTICs rely on PLK1 for survival because the PLK1 inhibitor BI2536 inhibited their growth in tumorsphere cultures. PLK1 inhibition suppressed growth, caused G(2) /M arrest, induced apoptosis, and reduced the expression of SOX2, a marker of neural stem cells, in SF188 cells. Consistent with SOX2 inhibition, the loss of PLK1 activity caused the cells to differentiate based on elevated levels of glial fibrillary acidic protein and changes in cellular morphology. We then knocked glial fibrillary acidic protein (GFAP) down SOX2 with siRNA and showed that it too inhibited cell growth and induced cell death. Likewise, in U251 cells, PLK1 inhibition suppressed cell growth, downregulated SOX2, and induced cell death. Furthermore, BI2536 delayed tumor growth of U251 cells in an orthotopic brain tumor model, demonstrating that the drug is active against GBM. In conclusion, PLK1 level is elevated in GBM and its inhibition restricts the growth of brain cancer cells.

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-RSK(S221/227) , P-YB-1(S102) , 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-RSK(S221/227) is present in primary TNBCs and correlates with P-YB-1(S102) as well as CD44. In conclusion, RSK2 inhibition provides a novel therapeutic avenue for TNBC and holds the promise of eliminating TICs.

Cold shock Y-box protein-1 proteolysis autoregulates its transcriptional activities.

BACKGROUND: The Y-box protein-1 (YB-1) fulfills pleiotropic functions relating to gene transcription, mRNA processing, and translation. It remains elusive how YB-1 shuttling into the nuclear and cytoplasmic compartments is regulated and whether limited proteolysis by the 20S proteasome releases fragments with distinct function(s) and subcellular distribution(s). RESULTS: To address these questions, mapping of domains responsible for subcellular targeting was performed. Three nuclear localization signals (NLS) were identified. NLS-1 (aa 149-156) and NLS-2 (aa 185-194) correspond to residues with unknown function(s), whereas NLS-3 (aa 276-292) matches with a designated multimerization domain. Nuclear export signal(s) were not identified. Endoproteolytic processing by the 20S proteasome before glycine 220 releases a carboxy-terminal fragment (CTF), which localized to the nucleus, indicating that NLS-3 is operative. Genotoxic stress induced proteolytic cleavage and nuclear translocation of the CTF. Co-expression of the CTF and full-length YB-1 resulted in an abrogated transcriptional activation of the MMP-2 promoter, indicating an autoregulatory inhibitory loop, whereas it fulfilled similar trans-repressive effects on the collagen type I promoter. CONCLUSION: Compartmentalization of YB-1 protein derivatives is controlled by distinct NLS, one of which targets a proteolytic cleavage product to the nucleus. We propose a model for an autoregulatory negative feedback loop that halts unlimited transcriptional activation.

Inhibition of p90 ribosomal S6 kinases disrupts melanoma cell growth and immune evasion.

BACKGROUND: The mitogen-activated protein kinase (MAPK) signaling pathway is frequently hyperactivated in malignant melanoma and its inhibition has proved to be an efficient treatment option for cases harboring BRAFV600 mutations (BRAFMut). However, there is still a significant need for effective targeted therapies for patients with other melanoma subgroups characterized by constitutive MAPK activation, such as tumors with NRAS or NF-1 alterations (NRASMut, NF-1LOF), as well as for patients with MAPK pathway inhibitor-resistant BRAFMut melanomas, which commonly exhibit a reactivation of this pathway. p90 ribosomal S6 kinases (RSKs) represent central effectors of MAPK signaling, regulating cell cycle progression and survival. METHODS: RSK activity and the functional effects of its inhibition by specific small molecule inhibitors were investigated in established melanoma cell lines and patient-derived short-term cultures from different MAPK pathway-hyperactivated genomic subgroups (NRASMut, BRAFMut, NF-1LOF). Real-time qPCR, immunoblots and flow cytometric cell surface staining were used to explore the molecular changes following RSK inhibition. The effect on melanoma cell growth was evaluated by various two- and three-dimensional in vitro assays as well as with melanoma xenograft mouse models. Co-cultures with gp100- or Melan-A-specific cytotoxic T cells were used to assess immunogenicity of melanoma cells and associated T-cell responses. RESULTS: In line with elevated activity of the MAPK/RSK signaling axis, growth and survival of not only BRAFMut but also NRASMut and NF-1LOF melanoma cells were significantly impaired by RSK inhibitors. Intriguingly, RSK inhibition was particularly effective in three-dimensional growth settings with long-term chronic drug exposure and suppressed tumor cell growth of in vivo melanoma models. Additionally, our study revealed that RSK inhibition simultaneously promoted differentiation and immunogenicity of the tumor cells leading to enhanced T-cell activation and melanoma cell killing. CONCLUSIONS: Collectively, RSK inhibitors exhibited both multi-layered anti-tumor efficacy and broad applicability across different genomic melanoma subgroups. RSK inhibition may therefore represent a promising novel therapeutic strategy for malignant melanoma with hyperactivated MAPK signaling.

Small interfering RNA library screen identified polo-like kinase-1 (PLK1) as a potential therapeutic target for breast cancer that uniquely eliminates tumor-initiating cells.

INTRODUCTION: Triple-negative breast cancer (TNBC) high rate of relapse is thought to be due to the presence of tumor-initiating cells (TICs), molecularly defined as being CD44high/CD24-/low. TICs are resilient to chemotherapy and radiation. However, no currently accepted molecular target exists against TNBC and, moreover, TICs. Therefore, we sought the identification of kinase targets that inhibit TNBC growth and eliminate TICs. METHODS: A genome-wide human kinase small interfering RNA (siRNA) library (691 kinases) was screened against the TNBC cell line SUM149 for growth inhibition. Selected siRNAs were then tested on four different breast cancer cell lines to confirm the spectrum of activity. Their effect on the CD44high subpopulation and sorted CD44high/CD24-/low cells of SUM149 also was studied. Further studies were focused on polo-like kinase 1 (PLK1), including its expression in breast cancer cell lines, effect on the CD44high/CD24-/low TIC subpopulation, growth inhibition, mammosphere formation, and apoptosis, as well as the activity of the PLK1 inhibitor, BI 2536. RESULTS: Of the 85 kinases identified in the screen, 28 of them were further silenced by siRNAs on MDA-MB-231 (TNBC), BT474-M1 (ER+/HER2+, a metastatic variant), and HR5 (ER+/HER2+, a trastuzumab-resistant model) cells and showed a broad spectrum of growth inhibition. Importantly, 12 of 28 kinases also reduced the CD44high subpopulation compared with control in SUM149. Further tests of these 12 kinases directly on a sorted CD44high/CD24-/low TIC subpopulation of SUM149 cells confirmed their effect. Blocking PLK1 had the greatest growth inhibition on breast cancer cells and TICs by about 80% to 90% after 72 hours. PLK1 was universally expressed in breast cancer cell lines, representing all of the breast cancer subtypes, and was positively correlated to CD44. The PLK1 inhibitor BI 2536 showed similar effects on growth, mammosphere formation, and apoptosis as did PLK1 siRNAs. Finally, whereas paclitaxel, doxorubicin, and 5-fluorouracil enriched the CD44high/CD24-/low population compared with control in SUM149, subsequent treatment with BI 2536 killed the emergent population, suggesting that it could potentially be used to prevent relapse. CONCLUSION: Inhibiting PLK1 with siRNA or BI 2536 blocked growth of TNBCs including the CD44high/CD24-/low TIC subpopulation and mammosphere formation. Thus, PLK1 could be a potential therapeutic target for the treatment of TNBC as well as other subtypes of breast cancer.

Medulloblastoma stem cells: where development and cancer cross pathways.

Brain tumors are the leading cause of childhood cancer mortality, with medulloblastoma (MB) representing the most frequent malignant tumor. The recent molecular classification of MB has reconceptualized the heterogeneity that exists within pathological subtypes by giving context to the role of key developmental signaling pathways in MB pathogenesis. The identification of cancer stem cell (CSC) populations, termed brain tumor-initiating cells (BTICs), in MB has provided novel cellular targets for the study of these aberrantly activated signaling pathways, namely, Sonic hedgehog (Shh) and Wingless (Wnt), along with the identification of novel BTIC self-renewal pathways. In this review, we discuss recent evidence for the presence of a MB stem cell that drives tumorigenesis in this malignant childhood tumor. We focus on evidence from cerebellar development, the recent identification of BTICs, the presence of activated developmental signaling pathways in MB, the role of epigenetic stem cell regulatory mechanisms, and how these developmental and epigenetic pathways may be targeted for novel therapeutic options.

Stem cells in brain tumour development and therapy- two-sides of the same coin.

Primary brain tumours are difficult to manage clinically due to their abilities to invade adjacent tissue and infiltrate distant neuropil. These contribute to challenges in surgical management and also limit the effectiveness of radiotherapy. Despite initial responses to chemotherapy, most tumours become chemo-resistant, leading to relapse. Recent identification and isolation of brain cancer stem cells (BCSCs) have broadened our understanding of the molecular pathogenesis and potential Achilles' heel of brain tumours. BCSCs are thought to drive and propagate the tumour and therefore present an important target for further investigations. This review explores the history of the discovery of BCSCs and the evolving concept of "cancer stem cells" in neuro-oncology. We attempt to present a balanced view on the subject and also to update the readers on the molecular biology of BCSCs. Lastly, we outline the potential strategies to target BCSCs which will translate into specific and effective therapies for brain tumours.

Subcellular localization of activated AKT in estrogen receptor- and progesterone receptor-expressing breast cancers: potential clinical implications.

Activated v-AKT murine thymoma viral oncogene homolog 1 (AKT)/protein kinase B (PKB) kinase (pAKT) is localized to the plasma membrane, cytoplasm, and/or nucleus in 50% of cancers. The clinical importance of pAKT localization and the mechanism(s) controlling this compartmentalization are unknown. In this study, we examined nuclear and cytoplasmic phospho-AKT (pAKT) expression by immunohistochemistry in a breast cancer tissue microarray (n = 377) with approximately 15 years follow-up and integrated these data with the expression of estrogen receptor (ER)alpha, progesterone receptor (PR), and FOXA1. Nuclear localization of pAKT (nuclear-pAKT) was associated with long-term survival (P = 0.004). Within the ERalpha+/PR+ subgroup, patients with nuclear-pAKT positivity had better survival than nuclear-pAKT-negative patients (P < or = 0.05). The association of nuclear-pAKT with the ERalpha+/PR+ subgroup was validated in an independent cohort (n = 145). TCL1 family proteins regulate nuclear transport and/or activation of AKT. TCL1B is overexpressed in ERalpha-positive compared with ERalpha-negative breast cancers and in lung metastasis-free breast cancers. Therefore, we examined the possible control of TCL1 family member(s) expression by the estrogen:ERalpha pathway. Estradiol increased TCL1B expression and increased nuclear-pAKT levels in breast cancer cells; short- interfering RNA against TCL1B reduced nuclear-pAKT. Overexpression of nuclear-targeted AKT1 in MCF-7 cells increased cell proliferation without compromising sensitivity to the anti-estrogen, tamoxifen. These results suggest that subcellular localization of activated AKT plays a significant role in determining its function in breast cancer, which in part is dependent on TCL1B expression.

Targeting tumour-initiating cells to improve the cure rates for triple-negative breast cancer.

Tumour recurrence is one of the biggest challenges in breast cancer management because it affects 25-30% of women with breast cancer and the tumours are often incurable. Women with triple-negative breast cancer (TNBC--lacking expression of the oestrogen receptor, progesterone receptor and the receptor HER2/ERBB2) have the highest rates of early recurrence relative to other breast cancer subtypes. Early recurrence might be due to tumour-initiating cells (TICs), which are resistant to conventional therapies, can remain dormant and can subsequently give rise to secondary tumours. In breast cancer, TICs are identified by the cell-surface markers CD44+/CD24-/EpCAM+ and/or possess ALDH1 enzyme activity. This subpopulation has the ability to self-renew, grow as mammospheres and initiate tumour formation. Fuelling the problem of relapse is the fact that chemotherapy and radiation can induce or select for TICs; this was reported in preclinical models and more recently in women being treated for breast cancer. Thus, new therapeutic agents for TNBC are presently being sought to overcome this problem. Here we review the roles of receptor tyrosine kinases, signalling intermediates and transcription factors in sustaining the TIC subpopulation. Particular emphasis is placed on targeting these molecules in order to eliminate and/or prevent the induction of TICs and ultimately reduce the frequency of TNBC recurrence.

The dawn of targeted therapies for triple negative breast cancer (TNBC): a snapshot of investigational drugs in phase I and II trials.

INTRODUCTION: Triple negative breast cancer (TNBC) was once thought to be an insurmountable disease marked by a lack of targeted treatments. However, we are now witnessing the dawn of targeted therapies for TNBC in which progress has stemmed from an improved understanding of the components that make TNBC unique. The identification of biomarkers, such as BRCA1/2, PIK3CA and RSK2, have advanced the field remarkably and there is considerable interest in finding novel therapeutics for TNBC that offer durable clinical benefit with fewer adverse events. AREAS COVERED: We discuss phase I/II trials of new and emerging targeted therapies for TNBC, according to ClinicalTrials.gov up to June 2020. Although the emphasis is on ongoing and completed early phase trials, we also highlight pivotal studies that have led to the approval of new targeted classes of drugs for TNBC, with a focus on outcomes and common adverse events of each class of therapy. EXPERT OPINION: The way forward for TNBC treatment is through precision medicine. The use of novel agents matched with biomarkers to identify patients with the best chance of sustainable response offers new hope. We now have great potential for improving the outcomes for patients with TNBC.

Nuclear detection of Y-box protein-1 (YB-1) closely associates with progesterone receptor negativity and is a strong adverse survival factor in human breast cancer.

BACKGROUND: Y-box binding protein-1 (YB-1) is the prototypic member of the cold shock protein family that fulfills numerous cellular functions. In the nucleus YB-1 protein orchestrates transcription of proliferation-related genes, whereas in the cytoplasm it associates with mRNA and directs translation. In human tumor entities, such as breast, lung and prostate cancer, cellular YB-1 expression indicates poor clinical outcome, suggesting that YB-1 is an attractive marker to predict patients' prognosis and, potentially, is suitable to individualize treatment protocols. Given these predictive qualities of YB-1 detection we sought to establish a highly specific monoclonal antibody (Mab) for diagnostic testing and its characterization towards outcome prediction (relapse-free and overall survival). METHODS: Hybridoma cell generation was carried out with recombinant YB-1 protein as immunogen and Mab characterization was performed using immunoblotting and ELISA with recombinant and tagged YB-1 proteins, as well as immunohistochemistry of healthy and breast cancer specimens. Breast tumor tissue array staining results were analyzed for correlations with receptor expression and outcome parameters. RESULTS: YB-1-specific Mab F-E2G5 associates with conformational binding epitopes mapping to two domains within the N-terminal half of the protein and detects nuclear YB-1 protein by immunohistochemistry in paraffin-embedded breast cancer tissues. Prognostic evaluation of Mab F-E2G5 was performed by immunohistochemistry of a human breast cancer tissue microarray comprising 179 invasive breast cancers, 8 ductal carcinoma in situ and 37 normal breast tissue samples. Nuclear YB-1 detection in human breast cancer cells was associated with poor overall survival (p = 0.0046). We observed a close correlation between nuclear YB-1 detection and absence of progesterone receptor expression (p = 0.002), indicating that nuclear YB-1 detection marks a specific subgroup of breast cancer. Likely due to limitation of sample size Cox regression models failed to demonstrate significance for nuclear YB-1 detection as independent prognostic marker. CONCLUSION: Monoclonal YB-1 antibody F-E2G5 should be of great value for prospective studies to validate YB-1 as a novel biomarker suitable to optimize breast cancer treatment.

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

INTRODUCTION: Basal-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. METHODS: Inhibition 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. RESULTS: Using 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, PKC alpha. 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. CONCLUSIONS: We 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.