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Supplementary Tables 1-6. Supplementary Table 1: Clinico-pathological demographics of CD133high and CD133low GBM; Supplementary Table 2: Positive and negative correlation probe sets present in CD133-signature; Supplementary Table 3: Drug targets acquired from CD133 gene signature connectivity map; Supplementary Table 4: qRT-PCR Primers; Supplementary Table 5: Genes used to complete network analysis; Supplementary Table 6: Molecular Subtypes of GBM samples used

Supplementary Figures 1-6. Supplementary Figure 1: CD133 correlates patient outcome with GIC selfrenewal and proliferative capacities in human GBM; Supplementary Figure 2: Gene Set Enrichment Analysis (GSEA) results for embryonic stem cell gene sets in CD133high- and CD133low-signature REMBRANDT samples; Supplementary Figure 3: CD133+ GICs are resistant to most compounds selected from CD133 connectivity mapping; Supplementary Figure 4: Pyrvinium specifically targets CD133+ GICs; Supplementary Figure 5: Overexpression of CD133; Supplementary Figure 6: Gene Set Enrichment Analysis (GSEA) results of Pyrvinium or DMSO control treated GICs

Purpose: Clonal evolution of cancer may be regulated by determinants of stemness, specifically self-renewal, and current therapies have not considered how genetic perturbations or properties of stemness affect such functional processes. Glioblastoma-initiating cells (GICs), identified by expression of the cell surface marker CD133, are shown to be chemoradioresistant. In the current study, we sought to elucidate the functional role of CD133 in self-renewal and identify compounds that can specifically target this CD133+ treatment-refractory population.Experimental Design: Using gain/loss-of-function studies for CD133 we assessed the in vitro self-renewal and in vivo tumor formation capabilities of patient-derived glioblastoma cells. We generated a CD133 signature combined with an in silico screen to find compounds that target GICs. Self-renewal and proliferation assays on CD133-sorted samples were performed to identify the preferential action of hit compounds. In vivo efficacy of the lead compound pyrvinium was assessed in intracranial GIC xenografts and survival studies. Lastly, microarray analysis was performed on pyrvinium-treated GICs to discover core signaling events involved.Results: We discovered pyrvinium, a small-molecule inhibitor of GIC self-renewal in vitro and in vivo, in part through inhibition of Wnt/β-catenin signaling and other essential stem cell regulatory pathways. We provide a therapeutically tractable strategy to target self-renewing, chemoradioresistant, and functionally important CD133+ stem cells that drive glioblastoma relapse and mortality.Conclusions: Our study provides an integrated approach for the eradication of clonal populations responsible for cancer progression, and may apply to other aggressive and heterogeneous cancers. Clin Cancer Res; 21(23); 5324–37. ©2015 AACR.

This review of the accounting education literature includes 101 articles published during 2018 in five journals: (1) Journal of Accounting Education, (2) Accounting Education, (3) Advances in Accounting Education: Teaching and Curriculum Innovations, (4) Issues in Accounting Education, and (5) The Accounting Educators’ Journal. We update 13 prior accounting education literature reviews by organizing and summarizing contributions to the accounting education literature made during 2018. Articles are categorized into five sections corresponding to traditional knowledge bases: (1) curriculum and instruction, (2) instruction by content area, (3) educational technology, (4) students, and (5) faculty. Research rigor of the empirical articles is discussed and critiqued. Suggestions for research in all areas are presented. Articles classified as instructional resources and cases published in the same five journals during 2018 are listed in appendices categorized by the relevant content area.

Background Low success rates in oncology drug development are prompting re-evaluation of preclinical models, including orthotopic tumor engraftment. In breast cancer models, tumor cells are typically injected into mouse mammary fat pads (MFP). However, this approach bypasses the epithelial microenvironment, potentially altering tumor properties in ways that affect translational application. Materials and methods Tumors were generated by mammary intraductal (MIND) engraftment of 4T1 carcinoma cells. Growth, histopathology, and molecular features were quantified. Results Despite growth similar to that of 4T1 MFP tumors, 4T1 MIND tumors exhibit distinct histopathology and increased metastasis. Furthermore, >6,000 transcripts were found to be uniquely up-regulated in 4T1 MIND tumor cells, including genes that drive several cancer hallmarks, in addition to two known therapeutic targets that were not up-regulated in 4T1 MFP tumor cells. Conclusion Engraftment into the epithelial microenvironment generates tumors that more closely recapitulate the complexity of malignancy, suggesting that intraductal adaptation of orthotopic mammary models may be an important step towards improving outcomes in preclinical drug screening and development.

Cigarette smoking is the main risk factor for chronic obstructive pulmonary disease, and to date, existing pharmacologic interventions have been ineffective at controlling inflammatory processes associated with the disease. To address this issue, we used the Connectivity Map (cMap) database to identify drug candidates with the potential to attenuate cigarette smoke-induced inflammation. We queried cMap using three independent in-house cohorts of healthy nonsmokers and smokers. Potential drug candidates were validated against four publicly available human datasets, as well as six independent datasets from cigarette smoke-exposed mice. Overall, these analyses yielded two potential drug candidates: kaempferol and bethanechol. Subsequently, the efficacy of each drug was validated in vivo in a model of cigarette smoke-induced inflammation. BALB/c mice were exposed to room air or cigarette smoke and treated with each of the two candidate drugs either prophylactically or therapeutically. We found that kaempferol, but not bethanechol, was able to reduce cigarette smoke-induced neutrophilia, both when administered prophylactically and when administered therapeutically. Mechanistically, kaempferol decreased expression of IL-1α and CXCL5 concentrations in the lung. Our data suggest that cMap analyses may serve as a useful tool to identify novel drug candidates against cigarette smoke-induced inflammation.

This review of the accounting education literature includes 103 articles published during 2017 in six journals: (1) Journal of Accounting Education, (2) Accounting Education, (3) Advances in Accounting Education: Teaching and Curriculum Innovations, (4) Global Perspectives on Accounting Education, (5) Issues in Accounting Education, and (6) The Accounting Educators’ Journal. We update 12 prior accounting education literature reviews by organizing and summarizing contributions to the accounting education literature made during 2017. Articles are categorized into five sections corresponding to traditional knowledge bases: (1) curriculum and instruction, (2) instruction by content area, (3) educational technology, (4) students, and (5) faculty. Research rigor of the empirical articles is discussed and critiqued. Suggestions for research in all areas are presented. Articles classified as instructional resources and cases published in the same six journals during 2017 are listed in appendices categorized by the relevant content area.

We analyzed publication trends in six accounting education journals published during the 20-year period 1997–2016: (1) Accounting Education, (2) Advances in Accounting Education, (3) Global Perspectives on Accounting Education, (4) Issues in Accounting Education, (5) Journal of Accounting Education, and (6) The Accounting Educators’ Journal. Our objective is to inform academicians about the overall development of accounting education research, empirical article topical trends, the extant knowledge base, and directions for future accounting education scholarship. Our analysis shows that the number of articles per year has increased over time and that the proportion of articles in four categories (empirical, descriptive, instructional resources, and cases) has changed over the 20-year period. The body of literature we analyzed shows the beginnings of a knowledge base that can be used as the foundation for future accounting education research.

Brain metastases (BM) are the most common brain tumor in adults and are a leading cause of cancer mortality. Metastatic lesions contain subclones derived from their primary lesion, yet their functional characterization is limited by a paucity of preclinical models accurately recapitulating the metastatic cascade, emphasizing the need for a novel approach to BM and their treatment. We identified a unique subset of stem-like cells from primary human patient brain metastases, termed brain metastasis-initiating cells (BMICs). We now establish a BMIC patient-derived xenotransplantation (PDXT) model as an investigative tool to comprehensively interrogate human BM. Using both in vitro and in vivo RNA interference screens of these BMIC models, we identified SPOCK1 and TWIST2 as essential BMIC regulators. SPOCK1 in particular is a novel regulator of BMIC self-renewal, modulating tumor initiation and metastasis from the lung to the brain. A prospective cohort of primary lung cancer specimens showed that SPOCK1 was overexpressed only in patients who ultimately developed BM. Protein-protein interaction network mapping between SPOCK1 and TWIST2 identified novel pathway interactors with significant prognostic value in lung cancer patients. Of these genes, INHBA, a TGF-β ligand found mutated in lung adenocarcinoma, showed reduced expression in BMICs with knockdown of SPOCK1. In conclusion, we have developed a useful preclinical model of BM, which has served to identify novel putative BMIC regulators, presenting potential therapeutic targets that block the metastatic process, and transform a uniformly fatal systemic disease into a locally controlled and eminently more treatable one.

This review of the accounting education literature includes 108 articles published during 2016 in six journals: (1) Journal of Accounting Education, (2) Accounting Education, (3) Advances in Accounting Education, (4) Global Perspectives on Accounting Education, (5) Issues in Accounting Education, and (6) The Accounting Educators’ Journal. This article updates prior accounting education literature reviews by organizing and summarizing recent contributions to the accounting education literature. Articles are categorized into five sections corresponding to traditional lines of inquiry: (1) curriculum and instruction, (2) instruction by content area, (3) educational technology, (4) students, and (5) faculty. Suggestions for research in all areas are presented. Articles presenting instructional resources and cases published in the same six journals during 2016 are listed in appendices categorized by the appropriate content area.

Background Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR). Methods We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line. Results We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551. Conclusions Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.

BackgroundBreast tumor growth and recurrence are driven by an infrequent population of breast tumor-initiating cells (BTIC). We and others have reported that the frequency of BTIC is orders of magnitude higher when breast tumor cells are propagated in vitro as clonal spheres, termed tumorspheres, by comparison to adherent cells. We exploited the latter to screen > 35,000 small molecules to identify agents capable of targeting BTIC. We unexpectedly discovered that selective antagonists of serotonin signaling were among the hit compounds. To better understand the relationship between serotonin and BTIC we expanded our analysis to include monoamine oxidase-A (MAO-A), an enzyme that metabolizes serotonin.MethodsWe used the Nanostring technology and Western blotting to determine whether MAO-A is expressed in human breast tumor cell lines cultured as tumorspheres by comparison to those grown as adherent cells. We then determined whether MAO-A activity is required for tumorsphere formation, a surrogate in vitro assay for BTIC, by assessing whether selective MAO-A inhibitors affect the frequency of tumorsphere-forming cells. To learn whether MAO-A expression in breast tumor cells is associated with other reported properties of BTIC such as anticancer drug resistance or breast tumor recurrence, we performed differential gene expression analyses using publicly available transcriptomic datasets.ResultsTumorspheres derived from human breast tumor cell lines representative of every breast cancer clinical subtype displayed increased expression of MAO-A transcripts and protein by comparison to adherent cells. Surprisingly, inhibition of MAO-A activity with selective inhibitors reduced the frequency of tumorsphere-forming cells. We also found that increased MAO-A expression is a common feature of human breast tumor cell lines that have acquired anticancer drug resistance and is associated with poor recurrence-free survival (RFS) in patients that experienced high-grade, ER-negative (ER−) breast tumors.ConclusionsOur data suggests that MAO-A activity is required for tumorsphere formation and that its expression in breast tumor cells is associated with BTIC-related properties. The discovery that a selective MAO-A inhibitor targets tumorsphere-forming cells with potencies in the nanomolar range provides the first evidence of this agent’s anticancer property. These data warrant further investigation of the link between MAO-A and BTIC.

This review of the accounting education literature includes 88 articles published during 2020 in five accounting education journals: (1) Journal of Accounting Education, (2) Accounting Education, (3) Advances in Accounting Education: Teaching and Curriculum Innovations, (4) Issues in Accounting Education, and (5) The Accounting Educators’ Journal. We update 15 prior accounting education literature reviews by organizing and summarizing contributions to the accounting education literature made during 2020. Articles are categorized into five sections corresponding to traditional knowledge bases: (1) curriculum and instruction, (2) instruction by content area, (3) educational technology, (4) students, and (5) faculty. We summarize the research design of the empirical articles. Suggestions for research are presented. Articles classified as cases published in the same five journals during 2020 are tabulated in an appendix categorized by content area.

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by functional sphere-forming assays. Consistent with these findings, human breast tumor cells express TPH1, 5-HT and SERT independent of their molecular or clinical subtype. Exposure of breast tumor cells ex vivo to sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), reduced BTIC frequency as determined by transplanting drug-treated tumor cells into immune-compromised mice. Moreover, another SSRI (vilazodone; Viibryd) synergized with chemotherapy to shrink breast tumor xenografts in immune-compromised mice by inhibiting tumor cell proliferation and inducing their apoptosis. Collectively our data suggest that antidepressants in combination with cytotoxic anticancer therapies may be an appropriate treatment regimen for testing in clinical trials.

Background: Triple Negative Breast Cancer (TNBC) is often associated with a poor prognosis. However TNBC is a heterogeneous group of tumors and while some patients have a poor prognosis others appear to do well long-term. There are currently no clinical or pathologic tumor features that distinguish poor from good outcome. Some TNBCs have infiltration with immune cells and the degree of this infiltration correlates with prognosis. Objectives: 1) To comprehensively examine immune factors associated with outcome in a cohort of TNBC patients, and 2) to develop an immune gene signature that can stratify patients into low and high risk groups. Methods: We profiled RNA from 22 TNBCs (10 who had experienced a recurrence) from our institutional cohort using the PanCancer Immune Profiling Panel from NanoString. This panel consists of an extensive list of 770 genes designed to evaluate the immune microenvironment of tumors. The genes fall into a number of functional categories including; 1. Genes that identify specific immune cells, 2. Cytokines that promote an effective immune response and others that are associated with immunosuppression, 3. Chemokines, which attract immune cells into the tumor, 4. Genes that assess both the activation and inhibition of immune cell function, 5. Genes that identify tumor specific antigens. Analysis was performed in the nSolver Advanced Analysis Program. Results: Using unsupervised hierarchical clustering of genes that were highly differentially expressed, the tumors were classified into 3 immune groups with distinct clinical outcomes. Group 1 ('Immune Excluded'), consisted of tumors with the lowest levels of expression of the immune markers assessed, suggesting that these tumors have a low or absent immune infiltrate; 7 of 7 patients in this group recurred. Group 2 ('Immune Activated') contains tumors that had the highest levels of anti-tumoral immune cell genes and their activation markers. This we interpret to represent a tumor group with a robust anti-tumor immune response; 0 of the 6 patients in this group recurred. In comparison Group 3 ('Immune Low') had moderate to low levels of expression of the majority of immune genes assessed. This we interpret to represent a group of tumors with limited immune cells present; 3 of 9 patients in this group recurred. Lastly, when comparing scores for immune cells, patients that recurred had lower scores for cytotoxic cells, CD8 T cells, Th1 cells and B cells. Conclusion: In this pilot study high expression of anti-tumoral immune genes correlated with good outcome, whereas lower/absent expression of these genes correlated with poor outcome. We are currently extending these findings to our entire cohort of 180 TNBC patients. Citation Format: Gillgrass AE, Pond GR, Levine MN, Whelan TJ, Hassell JA, Bane AL. The immune response in triple negative breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P4-12-09.

Medulloblastoma (MB), the most common malignant paediatric brain tumor, is currently treated using a combination of surgery, craniospinal radiotherapy and chemotherapy. Owing to MB stem cells (MBSCs), a subset of MB patients remains untreatable despite standard therapy. CD133 is used to identify MBSCs although its functional role in tumorigenesis has yet to be determined. In this work, we showed enrichment of CD133 in Group 3 MB is associated with increased rate of metastasis and poor clinical outcome. The signal transducers and activators of transcription-3 (STAT3) pathway are selectively activated in CD133+ MBSCs and promote tumorigenesis through regulation of c-MYC, a key genetic driver of Group 3 MB. We screened compound libraries for STAT3 inhibitors and treatment with the selected STAT3 inhibitors resulted in tumor size reduction in vivo. We propose that inhibition of STAT3 signaling in MBSCs may represent a potential therapeutic strategy to treat patients with recurrent MB.

This review of the accounting education literature includes 97 articles published during 2015 in six journals: (1) Journal of Accounting Education, (2) Accounting Education, (3) Advances in Accounting Education, (4) Global Perspectives on Accounting Education, (5) Issues in Accounting Education, and (6) The Accounting Educators' Journal. This article updates prior accounting education literature reviews by organizing and summarizing recent contributions to the accounting education literature. Articles are categorized into five sections corresponding to traditional lines of inquiry: (1) curriculum and instruction, (2) instruction by content area, (3) educational technology, (4) students, and (5) faculty. Suggestions for research in all areas are presented. Articles presenting instructional resources and cases published in the same six journals during 2015 are listed in appendices categorized by the appropriate content area.

Previous research has suggested that thyroid hormone receptor alpha 1 (THRα1), a hormone responsive splice variant, may play a role in breast cancer progression. Whether THRα1 can be exploited for anti-cancer therapy is unknown. The antiproliferative and antitumor effects of dronedarone, an FDA-approved anti-arrhythmic drug which has been shown to antagonize THRα1, was evaluated in breast cancer cell lines in vitro and in vivo. The THRα1 splice variant and the entire receptor, THRα, were also independently targeted using siRNA to determine the effect of target knockdown in vitro. In our study, dronedarone demonstrates cytotoxic effects in vitro and in vivo in breast cancer cell lines at doses and concentrations that may be clinically relevant. However, knockdown of either THRα1 or THRα did not cause substantial anti-proliferative or cytotoxic effects in vitro, nor did it alter the sensitivity to dronedarone. Thus, we conclude that dronedarone’s cytotoxic effect in breast cancer cell lines are independent of THRα or THRα1 antagonism. Further, the depletion of THRα or THRα1 does not affect cell viability or proliferation. Characterizing the mechanism of dronedarone’s anti-tumor action may facilitate drug repurposing or the development of new anti-cancer agents.

This review of the accounting education literature includes 256 articles published over the two-year period, 2013–2014, in six journals: (1) Journal of Accounting Education, (2) Accounting Education: An International Journal, (3) Advances in Accounting Education, (4) Global Perspectives on Accounting Education, (5) Issues in Accounting Education, and (6) The Accounting Educators' Journal. This article updates prior literature reviews by organizing and summarizing recent additions to the accounting education literature. These reviews are categorized into five sections corresponding to traditional lines of inquiry: (1) curriculum and instruction, (2) instruction by content area, (3) educational technology, (4) students, and (5) faculty. Suggestions for research in all areas are presented. Articles presenting instructional resources and cases published in the same six journals during 2013–2014 are listed in appendices categorized by the content area for which they are appropriate.

// Robin M. Hallett 1 , Jessica G. Cockburn 2 , Brian Li 2 , Anna Dvorkin-Gheva 1 , John A. Hassell 1 and Anita Bane 2 1 Department of Biochemistry and Biomedical Sciences, Centre for Functional Genomics, McMaster University, Hamilton, Ontario, Canada 2 Department of Oncology, McMaster University, Hamilton, Ontario, Canada Correspondence to: Anita Bane, email: // Keywords : basal-like breast cancer, gene expression, prognosis, networks Received : March 20, 2015 Accepted : April 07, 2015 Published : May 08, 2015 Abstract Purpose: Basal-like breast cancer (BLBC) is a molecular subtype of breast cancer associated with poor clinical outcome, although some patients with BLBC experience long-term survival. Apart from nodal status, current clinical/histopathological variables show little capacity to identify BLBC patients at either high- or low-risk of disease recurrence. Accordingly, we sought to develop a network based genomic predictor for predicting the outcome of patients with BLBC. Experimental Design: We performed network analysis on global gene expression profiling data of BLBCs, and identified BLBC network modules associated with AP-1 transcription, G-protein coupled receptors, and T-, B-, and NK-cells that are significant predictors of BLBC patient survival. Results: In gene expression and tissue microarray (TMA) validation cohorts of 210 and 102 BLBC patients, respectively, the identified network modules were robustly associated with patient outcome. In the gene expression validation cohort, the Kaplan-Meier estimate for 10-year survival in the low-risk group was 90%, whereas in the high-risk group it was a 56%. In the TMA cohort, the Kaplan-Meier estimate for 10-year survival in the low-risk group was 98%, whereas in the high-risk group it was 71%. Conclusions: The capacity to distinguish between patients with BLBC at high- or low-risk of recurrence at the time of diagnosis could permit timely intervention with more aggressive therapeutic regimens in those patients predicted to be high-risk, and to avoid such therapy in low-risk patients.

Background Natural killer (NK) cells play a critical role in cancer immunosurveillance. Recent developments in NK cell ex-vivo expansion makes it possible to generate millions of activated NK cells from a small volume of peripheral blood. We tested the functionality of ex vivo expanded NK cells in vitro against breast cancer cell lines and in vivo using a xenograft mouse model. The study aim was to assess functionality and phenotype of expanded NK cells from breast cancer patients against breast cancer cell lines and autologous primary tumours. Methods We used a well-established NK cell co-culture system to expand NK cells ex vivo from healthy donors and breast cancer patients and examined their surface marker expression. Moreover, we tested the ability of expanded NK cells to lyse the triple negative breast cancer and HER2-positive breast cancer cell lines MDA-MB-231 and MDA-MB-453, respectively. We also tested their ability to prevent tumour growth in vivo using a xenograft mouse model. Finally, we tested the cytotoxicity of expanded NK cells against autologous and allogeneic primary breast cancer tumours in vitro. Results After 3 weeks of culture we observed over 1000-fold expansion of NK cells isolated from either breast cancer patients or healthy donors. We also showed that the phenotype of expanded NK cells is comparable between those from healthy donors and cancer patients. Moreover, our results confirm the ability of ex vivo expanded NK cells to lyse tumour cell lines in vitro. While the cell lines examined had differential sensitivity to NK cell killing we found this was correlated with level of major histocompatibility complex (MHC) class I expression. In our in vivo model, NK cells prevented tumour establishment and growth in immunocompromised mice. Finally, we showed that NK cells expanded from the peripheral blood of breast cancer patients show high cytotoxicity against allogeneic and autologous patient-derived tumour cells in vitro. Conclusion NK cells from breast cancer patients can be expanded similarly to those from healthy donors, have a high cytotoxic ability against breast cancer cell lines and patient-derived tumour cells, and can be compatible with current cancer treatments to restore NK cell function in cancer patients.

Hematopoietic stem and progenitor cell differentiation are blocked in acute myeloid leukemia (AML) resulting in cytopenias and a high risk of death. Most patients with AML become resistant to treatment due to lack of effective cytotoxic and differentiation promoting compounds. High MN1 expression confers poor prognosis to AML patients and induces resistance to cytarabine and alltrans-retinoic acid (ATRA) induced differentiation. Using a high-throughput drug screening, we identified the dihydrofolate reductase (DHFR) antagonist pyrimethamine to be a potent inducer of apoptosis and differentiation in several murine and human leukemia cell lines. Oral pyrimethamine treatment was effective in two xenograft mouse models and specifically targeted leukemic cells in human AML cell lines and primary patient cells, while CD34+ cells from healthy donors were unaffected. The antileukemic effects of PMT could be partially rescued by excess folic acid, suggesting an oncogenic function of folate metabolism in AML. Thus, our study identifies pyrimethamine as a candidate drug that should be further evaluated in AML treatment.

Accumulating data suggests that the initiation and progression of human breast tumors is fueled by a rare subpopulation of tumor cells, termed breast tumor-initiating cells (BTIC), which resist radiotherapy and chemotherapy. Consequently, therapies that abrogate BTIC activity are needed to achieve durable cures for breast cancer patients. To identify such therapies we used a sensitive assay to complete a high-throughput screen of small molecules, including approved drugs, with BTIC-rich mouse mammary tumor cell populations. We found that inhibitors of the serotonin reuptake transporter (SERT) and serotonin receptors, which include approved drugs used to treat mood disorders, were potent inhibitors of mouse BTIC activity as determined by functional sphere-forming assays and the initiation of tumor formation by transplant of drug-exposed tumor cells into syngeneic mice. Moreover, sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), synergized with docetaxel (Taxotere) to shrink mouse breast tumors in vivo. Hence drugs targeting the serotonergic system might be repurposed to treat breast cancer patients to afford more durable breast cancer remissions.

Claudin-low breast cancer is a molecular type of breast cancer originally identified by gene expression profiling and reportedly associated with poor survival. Claudin-low tumors have been recognised to preferentially display a triple-negative phenotype, however only a minority of triple-negative breast cancers are claudin-low. We sought to identify an immunohistochemical profile for claudin-low tumors that could facilitate their identification in formalin fixed paraffin embedded tumor material. First, an in silico collection of ~1600 human breast cancer expression profiles was assembled and all claudin-low tumors identified. Second, genes differentially expressed between claudin-low tumors and all other molecular subtypes of breast cancer were identified. Third, a number of these top differentially expressed genes were tested using immunohistochemistry for expression in a diverse panel of breast cancer cell lines to determine their specificity for claudin-low tumors. Finally, the immunohistochemical panel found to be most characteristic of claudin-low tumors was examined in a cohort of 942 formalin fixed paraffin embedded human breast cancers with >10 years clinical follow-up to evaluate the clinico-pathologic and survival characteristics of this tumor subtype. Using this approach we determined that claudin-low breast cancer is typically negative for ER, PR, HER2, claudin 3, claudin 4, claudin 7 and E-cadherin. Claudin-low tumors identified with this immunohistochemical panel, were associated with young age of onset, higher tumor grade, larger tumor size, extensive lymphocytic infiltrate and a circumscribed tumor margin. Patients with claudin-low tumors had a worse overall survival when compared to patients with luminal A type breast cancer. Interestingly, claudin-low tumors were associated with a low local recurrence rate following breast conserving therapy. In conclusion, a limited panel of antibodies can facilitate the identification of claudin-low tumors. Furthermore, claudin-low tumors identified in this manner display similar clinical, pathologic and survival characteristics to claudin-low tumors identified from fresh frozen tumor material using gene expression profiling.

Background: Thyroid hormones promote breast cancer cell proliferation and expression of their cognate nuclear receptors has shown prognostic potential in small cohort studies. Among two isoforms of thyroid hormone receptor alpha (THRα), the alpha1 splice variant (THRα1) promotes thyroid hormone mediated cell proliferation whereas the alpha2 variant (THRα2) opposes it. Hence, THRα2 expression may be a favorable prognostic biomarker in breast cancer. Methods: A publicly available database of breast tumors archived by The Cancer Genome Atlas (TCGA) was employed for this study. We analyzed RNA expression of THRα1 and THRα2 in 106 triple negative breast cancers (TNBCs) and correlated it with tumor stage (I vs II vs III) and nodal involvement (positive vs negative). Tumor grade was not uniformly reported. Univariate Cox proportional hazards regression models were fitted to determine the prognostic impact of THRα1 and THRα2 expression on overall survival (OS) and multivariate models were adjusted for age, tumor stage and radiation treatment. Results: The median age of women was 54 (range 29-90) and 12.3% died. The majority (62.3%) of patients presented with stage II disease; 16.0% were stage III and 17.9% were stage I at diagnosis. There was no significant correlation between THRα1 or THRα2 expression and tumor stage or nodal involvement. Expression of THRα2 was associated with improved OS in both uni- and multi-variate models (Table). Conclusions: In this study, THRα2 expression was independently prognostic for improved OS in TNBC. We previously demonstrated similar results in 158 TNBCs via immunohistochemistry but differentiation between RNA (as opposed to protein) splice variants is more precise. These results support investigation of THRα2 up-regulation or THRα1 inhibition as therapeutic strategies. Table. Prognostic associations of THRα2 expression in TNBCVariableUnivariate HR (95%CI)p valueMultivariate HR (95%CI)p valueLog (THRα2)0.46 (0.26-0.81) Citation Format: Katarzyna J. Jerzak, Anna Dvorkin-Gheva, Jessica G. Cockburn, Anita Bane, John A. Hassell. Prognostic significance of thyroid hormone receptor-alpha-2 (THRα2) expression in triple-negative breast cancer: A TCGA study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2648.

Exposure of small animals to cigarette smoke is widely used as a model to study the pathogenesis of chronic obstructive pulmonary disease. However, protocols and exposure systems utilised vary substantially and it is unclear how these different systems compare. We analysed the gene expression profile of six publically available murine datasets from different cigarette smoke-exposure systems and related the gene signatures to three clinical cohorts. 234 genes significantly regulated by cigarette smoke in at least one model were used to construct a 55-gene network containing 17 clusters. Increasing numbers of differentially regulated clusters were associated with higher total particulate matter concentrations in the different datasets. Low total particulate matter-induced genes mainly related to xenobiotic/detoxification responses, while higher total particulate matter activated immune/inflammatory processes in addition to xenobiotic/detoxification responses. To translate these observations to the clinic, we analysed the regulation of the revealed network in three human cohorts. Similar to mice, we observed marked differences in the number of regulated clusters between the cohorts. These differences were not determined by pack-year. Although none of the experimental models exhibited a complete alignment with any of the human cohorts, some exposure systems showed higher resemblance. Thus, depending on the cohort, clinically observed changes in gene expression may be mirrored more closely by specific cigarette smoke exposure systems. This study emphasises the need for careful validation of animal models., Particulate matter skews gene expression pattern in cigarette smoke-exposed mice towards an inflammatory phenotype http://ow.ly/Tpex3038fyC

Brain Metastases (BM) are the most common type of cerebral tumor in adult, occurring at a rate 10 times greater than that of primary brain cancers. The inherent abilities of a primary tumor cell capable of initiating a BM resembles that of a cancer stem cell (CSCs). Previous work conducted in our lab identified a population of CSCs within lung-derived brain metastases. We hypothesize that a subgroup of CSCs, termed brain metastasis-initiating cells (BMICs), are responsible for the initiation of BM and are identifiable by an exclusive subset of genes that regulate self-renewal and metastasis. Despite the prevalence and lethality of BM, there is no clinically relevant model that fully reflects metastasis in patients. We recently generated a novel human-mouse xenotransplantation model of BM that allows for interrogation of each phase of the metastatic process from lung to brain, through injection of human patient-derived GFP-expressing BMICs into immunocompromised mice via three routes: 1) intracranial (IC), 2) intrathoracic (IT) and 3) intravascular/intracardiac (IV). GFP+ cells were harvested from the lungs and/or brains from each injection route, and RNA was submitted for microarray analysis to identify a unique metastatic and tissue-specific gene signature. In order to identify genes involved in self-renewal and tumor initiation, we also performed RNA interference screens in vitro and in vivo on BMIC lines against 150 genes implicated in BM formation. Further in vitro validation of select hit genes identified SPOCK1 and TWIST2 as essential regulators of cell proliferation, sphere formation, and migration. Future work will validate the role of these genes in our established in vivo metastasis models. We also aim to examine their expression in primary lung FFPE samples to determine if they are predictive biomarkers of lung-to-brain metastasis in prospective cohorts of newly diagnosed lung cancer patients, and to determine their potential as therapeutic targets.

Factors providing trophic support to diverse enteric neuron subtypes remain poorly understood. We tested the hypothesis that hepatocyte growth factor (HGF) and the HGF receptor MET might support some types of enteric neurons. HGF and MET are expressed in fetal and adult enteric nervous system. In vitro, HGF increased enteric neuron differentiation and neurite length, but only if vanishingly small amounts (1 pg/ml) of glial cell line-derived neurotrophic factor were included in culture media. HGF effects were blocked by phosphatidylinositol-3 kinase inhibitor and by MET-blocking antibody. Both of these inhibitors and MEK inhibition reduced neurite length. In adult mice, MET was restricted to a subset of calcitonin gene-related peptide-immunoreactive (IR) myenteric plexus neurons thought to be intrinsic primary afferent neurons (IPANs). Conditional MET kinase domain inactivation (Metfl/fl; Wnt1Cre+) caused a dramatic loss of myenteric plexus MET-IR neurites and 1–1′-dioctodecyl-3,3,3′,3′-tetramethylindocarbocyamine perchlorate (DiI) labeling suggested reduced MET-IR neurite length. In vitro, Metfl/fl; Wnt1Cre+ mouse bowel had markedly reduced peristalsis in response to mucosal deformation, but normal response to radial muscle stretch. However, whole-bowel transit, small-bowel transit, and colonic-bead expulsion were normal in Metfl/fl; Wnt1Cre+ mice. Finally, Metfl/fl; Wnt1Cre+ mice had more bowel injury and reduced epithelial cell proliferation compared with WT animals after dextran sodium sulfate treatment. These results suggest that HGF/MET signaling is important for development and function of a subset IPANs and that these cells regulate intestinal motility and epithelial cell proliferation in response to bowel injury. SIGNIFICANCE STATEMENT The enteric nervous system has many neuronal subtypes that coordinate and control intestinal activity. Trophic factors that support these neuron types and enhance neurite growth after fetal development are not well understood. We show that a subset of adult calcitonin gene-related peptide (CGRP)-expressing myenteric neurons produce MET, the receptor for hepatocyte growth factor, and that loss of MET activity affects peristalsis in response to mucosal stroking, reduces MET-immunoreactive neurites, and increases susceptibility to dextran sodium sulfate-induced bowel injury. These observations may be relevant for understanding and treating intestinal motility disorders and also suggest that enhancing the activity of MET-expressing CGRP neurons might be a useful strategy to reduce bowel inflammation.

Low oxygen tension (hypoxia) is a common characteristic of solid tumors and strongly correlates with poor prognosis and resistance to treatment. In response to hypoxia, cells initiate a cascade of transcriptional events regulated by the hypoxia inducible factor-1 (HIF-1) heterodimer. Since the oxygen-sensitive HIF-1α subunit is stabilized during hypoxia, it functions as the regulatory subunit of the protein. To date, while the mechanisms governing HIF-1α protein stabilization and function have been well studied, those governing HIF1A gene expression are not fully understood. However, recent studies have suggested that methylation of a HIF-1 binding site in the HIF1A promoter prevents its autoregulation. Here we report that the POZ-ZF transcription factor Kaiso modulates HIF1A gene expression by binding to the methylated HIF1A promoter in a region proximal to the autoregulatory HIF-1 binding site. Interestingly, Kaiso's regulation of HIF1A occurs primarily during hypoxia, which is consistent with the finding that Kaiso protein levels peak after 4 h of hypoxic incubation and return to normoxic levels after 24 h. Our data thus support a role for Kaiso in fine-tuning HIF1A gene expression after extended periods of hypoxia.

Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with average disease relapse at 9 months and median survival rarely extending beyond 15 months. Brain tumor stem cells (BTSCs) have been implicated in not only initiating GBM but also conferring resistance to therapy. However, it is not clear whether the BTSC population that initiates tumor growth is also responsible for GBM recurrence. In this study, we have developed a novel in vitro treatment model to profile the evolution of primary treatment-naive GBM BTSCs through chemoradiotherapy. We report that our in vitro model enriched for a CD15+/CD133- BTSC population, mirroring the phenotype of BTSCs in recurrent GBM. We also show that in vitro treatment increased stem cell gene expression as well as self-renewal capacity of primary GBMs. In addition, the chemoradiotherapy-refractory gene signature obtained from gene expression profiling identified a hyper-aggressive subtype of glioma. The delivery of in vitro chemoradiotherapy to primary GBM BTSCs models several aspects of recurrent GBM biology, and could be used as a discovery and drug-screening platform to uncover new biological drivers and therapeutic targets in GBM.

Brain Metastases (BM) represent a leading cause of cancer mortality. While metastatic lesions contain subclones derived from their primary lesion, their functional characterization has been limited by a paucity of preclinical models accurately recapitulating the stages of metastasis. This work describes the isolation of a unique subset of metastatic stem-like cells from primary human patient samples of BM, termed brain metastasis initiating cells (BMICs). Utilizing these BMICs we have established a novel patient-derived xenograft (PDX) model of BM that recapitulates the entire metastatic cascade, from primary tumor initiation to micro-metastasis and macro-metastasis formation in the brain. We then comprehensively interrogated human BM to identify genetic regulators of BMICs using in vitro and in vivo RNA interference screens, and validated hits using both our novel PDX model as well as primary clinical BM specimens. We identified SPOCK1 and TWIST2 as novel BMIC regulators, where in our model SPOCK1 regulated BMIC self-renewal and tumor initiation, and TWIST2 specifically regulated cell migration from lung to brain. A prospective cohort of primary lung cancer specimens was used to establish that SPOCK1 and TWIST2 were only expressed in patients who ultimately developed BM, thus establishing both clinical and functional utility for these gene products. This work offers the first comprehensive preclinical model of human brain metastasis for further characterization of therapeutic targets, identification of predictive biomarkers, and subsequent prophylactic treatment of patients most likely to develop BM. By blocking this process, metastatic lung cancer would effectively become a localized, more manageable disease.

The over treatment of prostate cancer patients is a significant concern, as recent clinical trials suggest that many patients are over treated which can lead to significant patient morbidity. Although the Gleason score is a powerful predictor of lethal or indolent disease, a significant proportion (∼40%) of men present with early stage Gleason score (GS) 7 tumors, for whom prognosis is variable. The goal of this study is to develop and optimize a robust prognostic gene signature that can be utilized on formalin fixed paraffin embedded (FFPE) core biopsy tumor material to better classify patients with intermediate risk, GS 7 tumors into good and poor outcome groups. Three gene signatures were derived from publicly available gene expression profiles of the Swedish Watchful Waiting cohort. The Genomic Grade Index consisted of the top 25 molecular signatures discriminating between high (8, 9 & 10) and low (≤ 6) GS tumors. The Lethal Gene Score consisted of the top 25 molecular signatures discriminating between lethal and indolent disease within GS 7 tumors only. A network-based gene signature consisted of 88 genes which accurately stratified GS 7 patients into high risk and low risk groups, resembling the survival curves of high GS and low GS patients. The prognostic capacity of the combined gene signature was tested in silico on the gene expression profiles of the Mayo cohort. Results demonstrated the gene signature's highly robust capacity for differentiating low risk and high risk patients within GS 7 patients. The NanoString nCounter System will be used to quantify mRNA from prostate FFPE blocks to assess the expression of the 138 prognostic genes. 156 archived prostate tumor blocks will be collected from intermediate risk, GS 7 patients enrolled in the 2005 PR5 prostate trial, which also collected 12 years of clinical follow-up information. Results will be correlated with biochemical (PSA) failure rates and overall survival. In short, our findings provide proof-of-principle that through the use of gene signatures it is possible to separate prostate cancer patients of intermediate risk into good and poor outcome groups. Furthermore, they also identify multiple gene candidates whose expression could likely be formulated into a clinically applicable assay, the implementation of which could serve to stratify prostate cancer patients with tumors of intermediate risk into more accurate high and low risk groups. Citation Format: Brian Li, Robin Hallet, Ying Wu, Greg Pong, John Hassell, Sebastien Hotte, Mark Levine, Himansu Lukka, Anita Bane. Prognostic gene signature for intermediate risk prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4346. doi:10.1158/1538-7445.AM2015-4346

e12082 Background: Thyroid hormones (THs) and their associated receptors may play a role in breast cancer progression given their promotion of cell growth and proliferation. THRα2 expression in ER ...

Medulloblastoma (MB) represents the most frequent malignant pediatric brain tumor. Since MBs characterized by activated sonic hedgehog (Shh) signaling continue to pose significant challenges due to treatment resistance, we aimed to prospectively identify, characterize, and target treatment-refractory Shh-dependent MB stem cells. Using gene expression and clinical outcome data from 325 primary human MBs, we identified Sox2high patients to exclusively represent treatment-refractory Shh MB. In order to prospectively characterize Sox2+ MB stem cells, we took advantage of the CRISPR-Cas9-nickase system to knock-in a P2A-mAG sequence into the Sox2 locus. By using our Sox2 reporter we show enchanced and reduced Sox2 expression in response to small molecule Shh agonists and inhibitors, respectively. Subsequent co-enrichment of Gli1/2 and H3K4me3 at the Sox2 promoter confirmed Sox2 as a downstream target in MB. In order to establish the clinical significance of this mechanism, we treated xenografts with a modified COG ACNS0332 radio/chemo-therapy protocol and prospectively tracked the in vivo evolution of Sox2+ cells. Molecular and functional analyses following treatment yielded an increase in Sox2 expression and self-renewal potential. Given the excellent clinical outcome in Wnt-driven MB patients, we aimed to convert treatment-resistant Sox2high MB into an ostensibly benign tumor using small molecule Wnt agonists. Wnt treatment resulted in a xenograft survival advantage and complete eradication of Sox2+ MB cells. Our work demonstrates mechanistic insight into the regulation of Sox2 by the Shh pathway and the clinical importance of this interaction by identifying Sox2 as a marker of treatment-resistant Shh MB stem cells. Our treatment model has enabled us to prospectively track the evolution of Sox2+ cells in vivo and thereby target these cells upon relapse. We have futher provided evidence for a novel therapeutic approach in converting aggressive MBs into a clinically-treatable tumor, which may improve the overall survival of children with treatment-refractory Shh-dependent MB.