The relationship between hypoxia, hypoxia gene signature and survival in patients with breast cancer

Thesis Summary Breast cancer is the most frequently diagnosed cancer in women in the UK. It is a heterogenous disease with subtypes which behave differently. Although several targeted therapies have been approved for patients with oestrogen receptor (ER) positive and Her-2 positive breast cancer, chemotherapy remains the standard systemic option for triple negative breast cancer (TNBC) patients. New prognostic tools for risk stratification and to guide use of the most aggressive treatments and new targeted therapies are desirable. The role of the tumour microenvironment (TME) in tumour progression is increasingly recognised. Features of the TME such as hypoxia have been reported to have a prognostic role in cancer. A better understanding of this feature may lead to identification of new prognostic and predictive tools and of new therapeutic targets for TNBC. The work of this thesis is carried out in three cohorts of patients with primary operable breast cancer and mature follow up. Data was available from clinical records regarding patient age, tumour pathology, treatment details and survival. Several hypoxic markers have been investigated and were reported to be overexpressed in breast cancer tissue. The aim of the current study was to investigate the role of hypoxia inducible factors [HIF1α (1), HIF-1α (2), HIF-2α] and carbonic anhydrases IX (CAIX) in different breast cancer subtypes, to establish biological processes, and key pathways related to cytoplasmic CAIX expression in ER-negative and a node negative subset of ER-negative breast cancer patients, and to identify the mRNA signature associated with CAIX within the tumour and stromal compartments in TNBC. Immunohistochemistry (IHC) was employed on a tissue microarray (TMA) of patients with breast cancer to assess the expression of HIF-1α (1), HIF-1α (2), HIF-2α and CAIX. Clinical outcomes for the marker’s expression were estimated using Kaplan-Meier analysis and compared between groups with the log-rank test. In a cohort of mixed breast cancer subtypes, the expression of cytoplasmic HIF-1α (1), and HIF-2α were only associated with poor overall survival (OS) in luminal A tumours (P = 0.009), and poor recurrence free survival (RFS) in Her-2 disease (P = 0.038), respectively. However, regardless of cellular localisation, high CAIX expression was associated with poor outcome for the full cohort and in breast cancer subtypes. High cytoplasmic CAIX was associated with decrease RFS in the full cohort (P<0.001), and in luminal B tumours (P = 0.025), disease-free survival (DFS) in the full cohort (P<0.001) in luminal B tumours (P = 0.035), and Her-2 disease (P = 0.016). Also, high cytoplasmic CAIX was associated with poor OS in the full cohort (P<0.001) and in Her-2 disease (P = 0.001). Moreover, membranous CAIX was associated with decrease RFS (P= 0.001), and OS (P = 0.003) in the full cohort. In multivariate analysis, cytoplasmic CAIX was an independent prognostic factor for RFS in the entire cohort (HR = 2.24, 95% CI: 1.19–4.22, P = 0.012), DFS in the full cohort (HR = 1.74, 95% CI: 1.08–2.82, P = 0.023) and in luminal B disease (HR = 3.59, 95% CI: 1.23–10.53, P = 0.020), and OS in Her-2 disease (HR = 4.19, 95% CI: 1.37–12.80, P = 0.012). Furthermore, in the cohort of ER-positive breast cancer, high cytoplasmic HIF-1α (1) expression was associated with shorter DFS (P = 0.032), and OS (P = 0.002) in the full cohort. In addition, high nuclear HIF-1α (1) expression was associated with decrease DFS in the full cohort (P = 0.009) and in luminal A disease (P = 0.013), and OS in the full cohort (P = 0.002) and in luminal A tumours (P = 0.003). Moreover, high cytoplasmic CAIX expression was corelated with worse RFS and DFS in the full cohort (P = 0.014, 0.008, respectively) and with RFS, DFS, and OS in luminal B disease (P = 0.018, 0.001, 0.003, respectively). On multivariate Cox regression analysis, nuclear HIF-1α (1) was an independent prognostic marker for DFS in the full cohort (HR = 1.85, 95% CI: 1.10–3.11, P = 0.019), and in luminal A disease (HR = 1.98, 95% CI: 1.02–3.83, P = 0.042), and for OS in the full cohort (HR = 1.85, 95% CI: 1.08–3.19, P = 0.026), and in luminal A disease (HR = 2.08, 95% CI: 1.11–3.89, P = 0.022). Moreover, high cytoplasmic CAIX expression was an independent prognostic marker for RFS and DFS in the full cohort (HR = 2.09, 95% CI: 1.17–3.75, P = 0.013; HR = 1.74, 95% CI: 1.08–2.82, P = 0.023), and in luminal B disease (HR = 2.57, 95% CI: 1.29–5.12, P = 0.007; HR = 2.75, 95% CI: 1.66–4.55, P<0.001), respectively. In TNBC cohort, high cytoplasmic expression of CAIX had lower RFS (P = 0.038). Multivariate analysis showed that cytoplasmic CAIX remained as factor contributing significantly to RFS (HR = 6.59, 95% CI: 1.47–29.58, P = 0.014). Next, Templated Oligo assay with Sequencing readout (TempO-Seq) (bulk RNAseq) was carried out in ER-negative and a node negative subset of ER-negative breast cancer patients to identify gene signatures that associated with CAIX expression to provide further information on biological processes, and key pathways related to cytoplasmic CAIX expression. Whole transcriptome analysis using TempO-Seq identified 10 significant genes within ER-negative cohort (OR8B2, SERHL2, KRT6A, MMP7, SPINK8, TMEM150C, CEACAM6, MUCL1, PITX2, and GALNT6), and 3 genes in node negative group (PCSK1N, SERHL2 and SPNS2). In node negative patients SPNS2 was of particular interest. Further, GeoMx Digital Spatial Profiler (DSP) analyses in TNBC cohort was performed to inform if gene signatures were associated with tumour epithelia or TME. 3 upregulated gene expression signatures, CD68, HIF1A, VSIR, and one down-regulated gene, pan-melanocyte, were identified in tumour compartment. In contrast, 9 downregulated genes, CD86, CD3E, MS4A1, BCL2, CCL5, NKG7, PTPRC, CD27 and FAS were identified in the TME in comparison of high and low CAIX expression groups. Among all 4 selected genes, HIF-1α, BCL2, CD68, and CD3, were further validated by IHC at protein level. Univariate KaplanMeier analysis showed high expression of CD68 and HIF-1α was associated with poor prognosis and high expression of BCL2 and CD3 was associated with good prognosis. By performing multivariate analysis for OS, high levels of CD68 cells in tumour nests and in TME were independent prognostic factor for poorer OS (HR = 2.42, 95% CI: 1.05–5.59, P = 0.038; HR = 3.34, 95% CI: 1.28–8.69, P = 0.014), respectively. In conclusion, this thesis has demonstrated a prognostic role of nuclear HIF-1α (1) and cytoplasmic CAIX in breast cancer. However, their prognostic values were different depending on cellular locations and tumour subtypes. Furthermore, TempO-Seq identified the pathways and genes associated with the CAIX in ER-negative breast cancer. Then, GeoMx DSP technology identified stromal/immune-related genes that were associated with TNBC patients’ survival in comparison of high and low CAIX expression groups that might serve as a potential prognostic biomarker for TNBC. Overall, the results from this thesis provide new evidence to warrant the further investigation of HIF-1α (1) and CAIX in a large contemporaneous cohort of patients with breast cancer and in particular in patients with TNBC

Epigenetic and Phenotypic Profile of Fibroblasts Derived from Induced Pluripotent Stem Cells

Human induced pluripotent stem (hiPS) cells offer a novel source of patient-specific cells for regenerative medicine. However, the biological potential of iPS-derived cells and their similarities to cells differentiated from human embryonic stem (hES) cells remain unclear. We derived fibroblast-like cells from two hiPS cell lines and show that their phenotypic properties and patterns of DNA methylation were similar to that of mature fibroblasts and to fibroblasts derived from hES cells. iPS-derived fibroblasts (iPDK) and their hES-derived counterparts (EDK) showed similar cell morphology throughout differentiation, and patterns of gene expression and cell surface markers were characteristic of mature fibroblasts. Array-based methylation analysis was performed for EDK, iPDK and their parental hES and iPS cell lines, and hierarchical clustering revealed that EDK and iPDK had closely-related methylation profiles. DNA methylation analysis of promoter regions associated with extracellular matrix (ECM)-production (COL1A1) by iPS- and hESC-derived fibroblasts and fibroblast lineage commitment (PDGFRβ), revealed promoter demethylation linked to their expression, and patterns of transcription and methylation of genes related to the functional properties of mature stromal cells were seen in both hiPS- and hES-derived fibroblasts. iPDK cells also showed functional properties analogous to those of hES-derived and mature fibroblasts, as seen by their capacity to direct the morphogenesis of engineered human skin equivalents. Characterization of the functional behavior of ES- and iPS-derived fibroblasts in engineered 3D tissues demonstrates the utility of this tissue platform to predict the capacity of iPS-derived cells before their therapeutic application

Continuity of the measure of the spectrum for quasiperiodic Schrodinger operators with rough potentials

We study discrete quasiperiodic Schr\"odinger operators on \ell^2(\zee) with potentials defined by $\gamma$-H\"older functions. We prove a general statement that for $\gamma >1/2$ and under the condition of positive Lyapunov exponents, measure of the spectrum at irrational frequencies is the limit of measures of spectra of periodic approximants. An important ingredient in our analysis is a general result on uniformity of the upper Lyapunov exponent of strictly ergodic cocycles.Comment: 15 page

Fibroblasts derived from human embryonic stem cells direct development and repair of 3D human skin equivalents

INTRODUCTION: Pluripotent, human stem cells hold tremendous promise as a source of progenitor and terminally differentiated cells for application in future regenerative therapies. However, such therapies will be dependent upon the development of novel approaches that can best assess tissue outcomes of pluripotent stem cell-derived cells and will be essential to better predict their safety and stability following in vivo transplantation. METHODS: In this study we used engineered, human skin equivalents (HSEs) as a platform to characterize fibroblasts that have been derived from human embryonic stem (hES) cell. We characterized the phenotype and the secretion profile of two distinct hES-derived cell lines with properties of mesenchymal cells (EDK and H9-MSC) and compared their biological potential upon induction of differentiation to bone and fat and following their incorporation into the stromal compartment of engineered, HSEs. RESULTS: While both EDK and H9-MSC cell lines exhibited similar morphology and mesenchymal cell marker expression, they demonstrated distinct functional properties when incorporated into the stromal compartment of HSEs. EDK cells displayed characteristics of dermal fibroblasts that could support epithelial tissue development and enable re-epithelialization of wounds generated using a 3D tissue model of cutaneous wound healing, which was linked to elevated production of hepatocyte growth factor (HGF). Lentiviral shRNA-mediated knockdown of HGF resulted in a dramatic decrease of HGF secretion from EDK cells that led to a marked reduction in their ability to promote keratinocyte proliferation and re-epithelialization of cutaneous wounds. In contrast, H9-MSCs demonstrated features of mesenchymal stem cells (MSC) but not those of dermal fibroblasts, as they underwent multilineage differentiation in monolayer culture, but were unable to support epithelial tissue development and repair and produced significantly lower levels of HGF. CONCLUSIONS: Our findings demonstrate that hES-derived cells could be directed to specified and alternative mesenchymal cell fates whose function could be distinguished in engineered HSEs. Characterization of hES-derived mesenchymal cells in 3D, engineered HSEs demonstrates the utility of this tissue platform to predict the functional properties of hES-derived fibroblasts before their therapeutic transplantation

Agent-based modeling of the impact of advertising on the regional economic cluster lifecycle

The aim of the study is the development and testing of an algorithm for modeling the impact of advertising on various stages of the life cycle of economic clusters. It is assumed, that the life cycle of the cluster consists of the stages: a diffuse group, a hidden cluster, an evolving cluster, a mature cluster, a collapsing cluster. Using the agent-based simulation methods, hierarchical clustering and chaos theory, the following results were obtained: a conceptual model of the behavior of cluster members for cluster formation processes at each stage of the cluster life cycle and an imitation model of the influence of advertising on the life cycle of the economic cluster; the patterns of various stages of the life cycle of the economic cluster and the functioning of the cluster without influence and under the influence of advertising were revealed. Advertising reduces the time at the stages of the associated life cycle of the cluster, increases the stage of maturity of the cluster. Companies that do not comply with the principles of clustering are under the influence of advertising and promotional activities. Such enterprises most often arise in the cluster at the stages of its formation

IA-CCF: Individual accountability for permissioned ledgers

Permissioned ledger systems allow a consortium of members that do not trust one another to execute transactions safely on a set of replicas. Such systems typically use Byzantine fault tolerance (BFT) protocols to distribute trust, which only ensures safety when fewer than 1/3 of the replicas misbehave. Providing guarantees beyond this threshold is a challenge: current systems assume that the ledger is corrupt and fail to identify misbehaving replicas or hold the members that operate them accountable—instead all members share the blame. We describe IA-CCF, a new permissioned ledger system that provides individual accountability. It can assign blame to the individual members that operate misbehaving replicas regardless of the number of misbehaving replicas or members. IA-CCF achieves this by signing and logging BFT protocol messages in the ledger, and by using Merkle trees to provide clients with succinct, universally-verifiable receipts as evidence of successful transaction execution. Anyone can audit the ledger against a set of receipts to discover inconsistencies and identify replicas that signed contradictory statements. IACCF also supports changes to consortium membership and replicas by tracking signing keys using a sub-ledger of governance transactions. IA-CCF provides strong disincentives to misbehavior with low overhead: it executes 47,000 tx/s while providing clients with receipts in two network round trips