Expression Of Therapeutic Targets Research Articles

Abstract P3-06-06: The importance of p300/CBP (CREBBP)-survivin (BIRC5) for cell cycle and apoptosis in triple negative breast cancer

Abstract In breast cancer, both incidence and mortality have been increasing. Triple negative breast cancer (TNBC), one of breast cancer subtypes, is a heterogeneous nature that is negative for estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor type 2 (HER2). Due to the lack of expression of therapeutic targets, new therapeutic strategy and elucidation the mechanism of action in TNBC are strongly desired. Histone acetyltransferases E1A binding protein (p300) and CREB binding protein (CBP) are coactivators of a large number of essential transcription factors that contribute to cell survival and proliferation. p300/CBP (CREBBP) interacts with transcription factors and other proteins through histone acetyltransferase (HAT) and regulates several gene expression. Dysregulation of CREBBP HAT activity could contribute to various diseases, including cancer. It is reported that the high expression of p300 correlates with poor prognosis in breast cancers. However, the mechanism of CREBBP HAT activity remains unclear. We, therefore, focused on CREBBP and clarified its important mechanism in TNBC. In breast cancer cell lines, p300 expression was higher than the normal mammary epithelial cell (MCF10A) in mRNA and protein levels and positively correlated with that of CBP. To evaluate the roles of p300 and CBP in cell survival and proliferation, the loss of function studies was conducted. In TNBC cell line (MDA-MB-231), cell survival and proliferation significantly reduced in p300 knockdown cells. The reduction in cell survival and proliferation was also observed using p300 inhibitor (L002). To examine the mechanism of action for the reduction of cell survival and proliferation in p300 inhibition, cell cycle assay and apoptosis assay were performed. We found that the cell-cycle progression was arrested at the cell cycle G2/M phase and apoptosis induction in p300 knockdown cells. These results suggest that cell cycle G2/M phase arrest and the late stage of apoptosis are caused by p300 inhibition and p300 inhibition enhances the anti-tumor effect. To identify a mechanism responsible for the reduction of cell survival and proliferation in p300 inhibition, antibody array was performed. As a result of this assay and TCGA patients cohorts analysis, we identified survivin (BIRC5), a member of the inhibitor of apoptosis (IAP) family, as a functional novel biomarker of TNBC with significantly higher expression. These results suggest that suppression of BIRC5 through CREBBP inhibitor result in G2/M phase cell cycle arrest and apoptotic cell death in vitro and in vivo. In conclusion, CREBBP-BIRC5 transcriptional complex could be a novel therapeutic target for TNBC. Citation Format: Sunao Tanaka, Natsuki Wariishi, Nobuko Kawaguchi-Sakita, Souichi Adachi, Masakazu Toi, Yasuhiko Kamikubo. The importance of p300/CBP (CREBBP)-survivin (BIRC5) for cell cycle and apoptosis in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-06-06.

Molecular profiling in muscle-invasive bladder cancer: more than the sum of its parts.

Bladder cancers are biologically and clinically heterogeneous. Recent large-scale transcriptomic profiling studies focusing on life-threatening muscle-invasive cases have demonstrated a small number of molecularly distinct clusters that largely explain their heterogeneity. Similar to breast cancer, these clusters reflect intrinsic urothelial cell-type differentiation programs, including those with luminal and basal cell characteristics. Also like breast cancer, each cell-based subtype demonstrates a distinct profile with regard to its prognosis and its expression of therapeutic targets. Indeed, a number of studies suggest subtype-specific differential responses to cytotoxic chemotherapy and to therapies that inhibit a number of targets, including growth factors (EGFR, ERBB2, FGFR) and immune checkpoint (PD1, PDL1) inhibitors. Despite burgeoning evidence for important clinical implications, subtyping has yet to enter into routine clinical practice. Here we review the conceptual basis for intrinsic cell subtyping in muscle-invasive bladder cancer and discuss evidence behind proposed clinical uses for subtyping as a prognostic or predictive test. In deliberating barriers to clinical implementation, we review pitfalls associated with transcriptomic profiling and illustrate a simple immunohistochemistry (IHC)-based subtyping algorithm that may serve as a faster, less expensive alternative. Envisioned as a research tool that can easily be translated into routine pathology workflow, IHC-based profiling has the potential to more rapidly establish the utility (or lack thereof) of cell type profiling in clinical practice. Copyright © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 3792: Improved isolation and detection of circulating tumor cells of pancreatic cancer patients with characterization by mutational analysis

Abstract Introduction: Isolation of circulating tumor cells based on cell surface markers is often hindered by epithelial to mesenchymal transition with loss of epithelial antigens. Low numbers of CTCs and fewer cases of CTC positive pancreatic cancer compared to other cancers (e.g. breast cancer) lead to the assumption that pancreatic tumors do not release CTCs as frequently or that the pancreatic CTCs lack EpCAM expression. Isolation of CTCs based on their size is independent of EMT-like phenotypical changes. We therefore compared a filtration-based isolation method with an EpCAM-based isolation method. We optimized CTC detection by using a highly sensitive anti-cytokeratin antibody panel for the detection of cancer cells with moderate cytokeratin expression. In order to use isolated CTCs as liquid biopsy for tumor characterization and treatment selection, downstream analysis is necessary. We here show the feasibility of mutational analysis of isolated CTCs by castPCR. Methods: EpCAM high, medium and low cells were enriched and isolated by an EpCAM-based immunomagnetic procedure (IsoFlux) and a filtration device (Siemens) to determine recovery rates of both methods. Isolated cells were detected by an improved immunofluorescent staining with an anti-Cytokeratins, anti-EpCAM and anti-PBMC panel and characterized by competitive allele-specific TaqMan PCR (castPCR) for KRAS mutations. Results: Cytokeratin expression is crucial for detection of CTCs in a high background of blood cells. Improvement of the staining protocol helps to increase the sensitivity of detection. The filtration based approach is superior to the surface antigen-based isolation. With the size-dependent method we obtained a recovery rate of 52 % even for EpCAM-low cells compared to only 1 % based on the immune-affinity purification. Cells isolated by filtration can be characterized for expression of therapeutic targets by immunostaining. The utility of the size dependent platform for subsequent functional characterization of the CTCs was also demonstrated by detection of k-ras mutations in single isolated CTCs by castPCR. Conclusion: For CTCs undergoing EMT, filtration yields higher recovery compared to the standard surface antigen (EpCAM)-based methods. Isolation by filtration also allows for mutational analysis which can be used to confirm the identity of the isolated cell as CTC. In addition, mutational analysis of CTCs can be used to guide the treatment of patients. Use of liquid biopsies for treatment selection will facilitate truly personalized medicine. Citation Format: Oliver von Ahsen, Nora Brychta, Thomas Krahn. Improved isolation and detection of circulating tumor cells of pancreatic cancer patients with characterization by mutational analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3792. doi:10.1158/1538-7445.AM2017-3792

Circulating and disseminated tumor cells: diagnostic tools and therapeutic targets in motion.

Enumeration of circulating tumor cells (CTCs) in peripheral blood with the gold standard CellSearchTM has proven prognostic value for tumor recurrence and progression of metastatic disease. Therefore, the further molecular characterization of isolated CTCs might have clinical relevance as liquid biopsy for therapeutic decision-making and to monitor disease progression. The direct analysis of systemic cancer appears particularly important in view of the known disparity in expression of therapeutic targets as well as epithelial-to-mesenchymal transition (EMT)-based heterogeneity between primary and systemic tumor cells, which all substantially complicate monitoring and therapeutic targeting at present. Since CTCs are the potential precursor cells of metastasis, their in-depth molecular profiling should also provide a useful resource for target discovery. The present review will discuss the use of systemically spread cancer cells as liquid biopsy and focus on potential target antigens.

Abstract A66: Systemic effects of AC-T chemotherapy in breast cancer

Abstract Cytotoxic chemotherapy is the only standard of care treatment currently available for patients with triple negative breast cancer (TNBC) because this breast cancer subtype lacks expression of therapeutic targets, namely, estrogen receptor, progesterone receptor, and amplification of Her2. Despite the efficacy of neoadjuvant chemotherapy for some TNBC patients, approximately 60-80% of TNBC patients will still present with local and distant disease after therapy, and these patients suffer poor prognosis. In addition to direct effects on tumor cells, systemic chemotherapy also has the potential to impact patient physiology. However, the implications of tumor-cell independent effects of chemotherapy for metastatic progression are not well understood. We are using mouse models of TNBC to study the effects of chemotherapy on host physiology and how these effects impact metastasis. Treatment of TNBC tumor-bearing mice with doxorubicin (A), cyclophosphamide (C), and paclitaxel (T) decreased overall bone marrow cell counts; although, the degree of decrease and subsequent recovery of individual bone marrow and circulating cell populations varied among populations. Notably, parallel experiments in tumor-free mice demonstrated that chemotherapy-dependent effects on the bone marrow and circulating cell populations dominate tumor-dependent effects. These findings highlight the potential implications of the physiological effects of chemotherapy on metastasis and response to therapy. Citation Format: Molly J. DeCristo, Sandra S. McAllister. Systemic effects of AC-T chemotherapy in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A66.

Abstract 415: Differential expression of therapeutic targets across tumor micro-environments and at infiltrative margins in glioblastoma

Abstract Glioblastoma (GBM; WHO grade IV) is the most common primary brain tumor and is associated with a poor prognosis. The Cancer Genome Atlas (TCGA) project has revealed discrete molecular classes of GBM, raising the possibility of targeted therapies. Molecular heterogeneity across tumor microenvironments presents a challenge to targeted therapy, since effective therapies will be directed at targets near resection margins rather than those expressed in bulk resected tumor. To this end, we initiated studies to delineate spatial and temporal variations of potential druggable targets in GBM, focusing on potential protein targets at the tumor margin. By utilizing 5-ALA (5-aminolevulinic acid, an oral compound that accumulates in malignant glioma cells and is metabolized to the fluorescent agent, protoporphyrin IX) fluorescence-guided surgery of primary and recurrent GBMs in a Phase II clinical trial, we were able to precisely define perinecrotic, bulk, and marginal regions within the glioma intraoperatively in order to sample tissues for further molecular characterization. Tissues harvested from these 3 tumor regions from 5 patients were analyzed using reverse phase protein array (RPPA) analysis with 218 antibodies (Array-Pro). Relative protein levels were determined and a heatmap was generated as a hierarchical cluster using Pearson Correlation and a center metric. Forty proteins had statistically significant altered expression at the tumor margin compared with bulk and perinecrotic tumor. Among these, STAT1, STAT2, STAT3, PDGFRA, PDGFRB, EGFR, JAK2, MET, PIK3R1, and ERBB2 were upregulated at the margins and are potential druggable targets worthy of further investigation. Also upregulated at the tumor margins were NFKBIA, JUN, ZAP70, GAPDH, and CD28, whereas STAT5A, EFNB2, NTRK1, KDR, FGFR1, PTPRA, IFNGR1, and EFNB1 were downregulated at margins. Our data demonstrate significant variation of druggable targets and signaling networks across tumor microenvironments in GBM. Marginal tumor differs from bulk and perinecrotic tumor by the relative overexpression of STAT proteins, growth factor receptors, and receptor kinases. Directing therapy at networks and targets present at the tumor margin of GBM may be an effective strategy. Citation Format: Myles R. McCrary, David Gutman, William Dunn, Milota Kaluzova, Alexandros Bouros, Merete Williams, Xialong Zhang, Lee AD Cooper, Constantinos G. Hadjipanayis, Daniel J. Brat. Differential expression of therapeutic targets across tumor micro-environments and at infiltrative margins in glioblastoma. [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 415. doi:10.1158/1538-7445.AM2015-415

Abstract 10: Comparison of outcomes in patients undergoing rectosigmoid resection: End colostomy versus reanastomosis

9: Identification of molecular markers for targeted treatment of uterine sarcoma J. Shank, J. Rhode, J. Liu. University of Michigan, Ann Arbor, MI, USA, Monarch Women's Cancer Center, Salt Lake City, UT, USA Objectives: Uterine sarcomas are rare and aggressive tumor types. Patients diagnosed with this disease often have poor clinical outcomes, despite multi-modal therapy. Because uterine sarcomas are not common, investigations into novel therapeutics for this disease have been limited. The aim of our study was to identify the expression of therapeutic targets using tissue microarrays in order to direct the exploration of molecularly targeted treatment. Methods: Tumor tissue was collected from 36 patients with uterine sarcomas (6 endometrial stromal sarcomas [ESS], 7 leiomyosarcomas [LMS], and 23 carcinosarcoma [CS]) who were evaluated at a single cancer center between 1996 and 2004. Tissue microarrays were constructed and expression of several molecular markers that are associated with targeted therapeutics in current use for other tumor types was assessed. Expression of VEGF, EGRF, c-Kit and HER-2 was investigated using immunohistochemistry. Immunostaining was evaluated by a gynecologic pathologist. Clinical outcomes including demographic data, treatment modalities received, and overall survival were recorded. Results: The mean age of patients included in this study was 63 (range 35–91). Expression of VEGF, EGFR, c-KIT, and HER-2 was detected in 81%, 81%, 72%, and 14% of all uterine sarcomas examined respectively. VEGF was expressed in 17% of ESS (1/6), 86% of LMS (6/ 7), and 96% of CS (22/23). EGFR was expressed in 50% of ESS (3/6), 86% of LMS (6/7), and 87% of CS (20/23). Expression of c-KIT was seen in 50% of ESS (3/6), 57% of LMS (4/7), and 83% of CS (19/23). HER-2 expression was minimal in all tumor types (ESS 17% [1/6], no expression in any LMS samples, and CS 7% [4/23]). Conclusions: Our data suggests that anti-angiogenic therapy may have activity in uterine leiomyosarcomas and carcinosarcomas. Targeted therapy for EGFR may have significant effects in patients with LMS and CS. This finding may justify using monoclonal antibodies against EGFR (such as cetuximab) or small molecule inhibitors of EGFR (such as gefitinib) in patients with LMS and CS. c-KIT also may be a potential target in CS and further studies with imatinib may be justified. HER-2 is a poor therapeutic target in all three types of uterine sarcomas. These results provide rationale for the use of targeted therapy to improve outcomes for uterine sarcoma

Abstract 265: Mechanisms of Met-dependent tumorigenesis in models of triple negative breast cancer.

Abstract Human breast cancer is a heterogeneous disease encompassing multiple subtypes. The basal-like and claudin-low subtypes, collectively known as triple negative (TN) breast cancer, are predominantly negative for the expression of therapeutic targets (Her2 and estrogen receptors) and have poor prognosis. In human breast cancer, elevated levels of MET are correlated with TN subtypes and poor outcome, but the reasons for this are relatively unknown. The Met receptor tyrosine kinase (RTK) is a cell surface protein that is activated in response to the Hepatocyte Growth Factor (HGF). Upon stimulation, Met signaling pathways initiate a program of cell survival, migration, and invasive growth. To investigate the role of Met in tumourigenesis, we generated transgenic mice with mammary specific expression of an oncogenic variant of Met (MMTV-Metmut). These mice develop tumours with features of TN breast cancer. Tumour-initiating cells (TICs) have emerged as a key concept in prevailing models of tumour development. In breast and other solid tumours, where the bulk of the tumour mass consists of differentiated non-tumourigenic cells, TICs constitute a cell fraction of less differentiated tumourigenic cells, and have been shown to be highly resistant to radiation and chemotherapy. TICs are thus a key therapeutic target, yet little is known about TICs in TN breast cancer. Using tumoursphere assays, we have established the presence of TICs in tumours derived from MMTV-Metmut transgenic mice. MMTV-Metmut TICs have constitutively active Met. Interestingly, treatment with a small molecule inhibitor targeting Met can diminish both tumoursphere formation and proliferation. Thus, in TN breast cancers, elevated levels of Met signaling may promote TIC tumourigenicity. Here, mechanisms of Met-dependent TIC propagation and tumourigenicity will be addressed and supporting data presented. The molecular events that lead to human basal-like and claudin-low breast cancers are poorly understood. Validating a role for Met in TICs could identify key pathways involved in the pathogenesis of TN breast cancer, providing information for new therapeutic targets. Citation Format: Vanessa Y.C. Sung, Jennifer F. Knight, Morag Park. Mechanisms of Met-dependent tumorigenesis in models of triple negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 265. doi:10.1158/1538-7445.AM2013-265

Expression of therapeutic targets in Ewing sarcoma family tumors

Ewing sarcoma family tumor is an aggressive malignant tumor of bone and soft tissue in children and adolescents. Despite advances in modern therapy, metastasis occurs in 20% to 25% of cases and results in mortality in 80% of patients. Intracellular molecules mammalian target of rapamycin, Akt, vascular endothelial growth factor, nuclear factor κB, and BRAF are important kinases and transcription factors that regulate the proliferation of tumor cells. We studied the expression of these proteins in 72 Ewing sarcoma family tumors. Patients' survival data were available in 55 cases. Formalin-fixed, paraffin-embedded tumor sections were stained with antibodies against phosphorylated mammalian target of rapamycin, Akt, BRAF, vascular endothelial growth factor, and nuclear factor κB proteins. Stained sections were analyzed for percentage and strength of staining, and a composite score (0-200) was subsequently generated. Although most tumors expressed mammalian target of rapamycin, Akt, nuclear factor κB, and vascular endothelial growth factor, only 37%, 86%, 55%, and 12%, respectively, showed high expression (staining score ≥ 100). There was no significant correlation between mammalian target of rapamycin and Akt expression and clinical outcome. High nuclear factor κB expression was significantly associated with tumors in pelvic locations. Decreased vascular endothelial growth factor expression (score <100) was significantly associated with better prognosis (P < .05). BRAF was not expressed in most cases and showed negative or weak staining (score <100) in 97% of cases. Thus, except for BRAF, Ewing sarcoma family tumors may be amenable to treatment that targets the expressed proteins. High Akt expression suggests potential universal response to Akt-targeted therapy. BRAF kinase inhibitors are unlikely to be effective in the treatment of Ewing sarcoma family tumors.

Abstract BSF1-1: Single Cell Analysis of Normal and Tumor Breast Epithelium Reveals Differential Use of Signalling Pathways by Novel Populations

Abstract Primary human tissue contains a complex mixture of cell types which can only be partially purified by cell sorting based on surface markers. To gain further insight into mammary gland and tumor biology, we performed highly parallel single cell multiplexed gene expression measurements on individual normal and malignant mammary cells. This approach enabled discovery of novel normal mammary stem cell markers and gave insights into normal and malignant tissue architecture. We identified two distinct normal mammary stem cell populations: a proliferating, stem cell population that expresses basal cell markers and shows evidence of activation of the Wnt/β-catenin signaling pathway components and a second, Axin2-, CD44-/low quiescent normal stem cell population which does not express basal cell markers. Both populations had similar engraftment ability in vivo, demonstrating that there are likely two physiological stem cell states. Single cell analysis of estrogen receptor positive (ER+) breast tumors revealed similar cellular populations in cancer cells that resemble the normal mammary duct architecture. Remarkably, the expression of therapeutic targets such as growth factor receptors and the estrogen receptor differed between the different cell compartments. Most notably, the Axin2- cell population did not express ER or TOPO2, the target of Adriamycin, one of the most effective drugs for the treatment of breast cancer, suggesting that this population is not directly targeted by many therapeutic agents routinely used for the treatment of ER+ breast cancer. Our results show that single cell analyses might be useful for developing new strategies for treating cancer by identifying the expression of therapeutic targets by each of the cancer cell populations that make up a particular tumor. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr BSF1-1.