A Novel Mathematical Approach for Analysis of Integrated Cell-Patient Data Uncovers a 6-Gene Signature Linked to Endocrine Therapy Resistance.

A significant number of breast cancers develop resistance to hormone therapy. This progression, while posing a major clinical challenge, is difficult to predict. Despite important contributions made by cell models and clinical studies to tackle this problem, both present limitations when taken individually. Experiments with cell models are highly reproducible but do not reflect the indubitable heterogenous landscape of breast cancer. On the other hand, clinical studies account for this complexity but introduce uncontrolled noise due to external factors. Here, we propose a new approach for biomarker discovery that is based on a combined analysis of sequencing data from controlled MCF7 cell experiments and heterogenous clinical samples that include clinical and sequencing information from The Cancer Genome Atlas. Using data from differential gene expression analysis and a Bayesian logistic regression model coupled with an original simulated annealing-type algorithm, we discovered a novel 6-gene signature for stratifying patient response to hormone therapy. The experimental observations and computational analysis built on independent cohorts indicated the superior predictive performance of this gene set over previously known signatures of similar scope. Together, these findings revealed a new gene signature to identify patients with breast cancer with an increased risk of developing resistance to endocrine therapy.

Intravital imaging of Wnt/ß-catenin and ATF2-dependent signalling pathways during tumour cell invasion and metastasis.

Wnt signalling has been implicated as a driver of tumour cell metastasis, but less is known about which branches of Wnt signalling are involved and when they act in the metastatic cascade. Here, using a unique intravital imaging platform and fluorescent reporters, we visualised ß-catenin/TCF-dependent and ATF2-dependent signalling activities during human cancer cell invasion, intravasation and metastatic lesion formation in the chick embryo host. We found that cancer cells readily shifted between states of low and high canonical Wnt activity. Cancer cells that displayed low Wnt canonical activity showed higher invasion and intravasation potential in primary tumours and in metastatic lesions. In contrast, cancer cells showing low ATF2-dependent activity were significantly less invasive both at the front of primary tumours and in metastatic lesions. Simultaneous visualisation of both these reporters using a double-reporter cell line confirmed their complementary activities in primary tumours and metastatic lesions. These findings might inform the development of therapies that target different branches of Wnt signalling at specific stages of metastasis.

Patient-Derived Explant Cultures of Normal and Tumor Human Breast Tissue.

Tissue culture has evolved considerably over the last few years, including cell culture in three dimensions, organoids, cocultures of different cell types and the use of diverse types of matrices in an attempt to mimic conditions that more closely resemble those found in the original tissue or organ. In this chapter, we describe how patient-derived breast tissue can be cultured on sponges for several days, maintaining their original architecture and with the capacity to respond to treatments. This protocol facilitates the study of the tissue responses without the need for extensive tissue manipulation, cell digestion or use of a biomaterial as scaffold, while maintaining the stroma and extracellular matrix organization. This method has the potential to improve preclinical testing by contributing to provide more accurate data reflecting cell-cell and cell-matrix interactions, tumor microenvironment, drug effects or stem cell function in normal- and pathophysiology of the breast.

The Tumor Suppressor ING5 Is a Dimeric, Bivalent Recognition Molecule of the Histone H3K4me3 Mark.

The INhibitor of Growth (ING) family of tumor suppressors regulates the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at lysine 4 (H3K4me3). This modification is recognized by the plant homeodomain (PHD) present at the C-terminus of the five ING proteins. ING5 facilitates histone H3 acetylation by the HBO1 complex, and also H4 acetylation by the MOZ/MORF complex. We show that ING5 forms homodimers through its N-terminal domain, which folds independently into an elongated coiled-coil structure. The central region of ING5, which contains the nuclear localization sequence, is flexible and disordered, but it binds dsDNA with micromolar affinity. NMR analysis of the full-length protein reveals that the two PHD fingers of the dimer are chemically equivalent and independent of the rest of the molecule, and they bind H3K4me3 in the same way as the isolated PHD. We have observed that ING5 can form heterodimers with the highly homologous ING4, and that two of three primary tumor-associated mutants in the N-terminal domain strongly destabilize the coiled-coil structure. They also affect cell proliferation and cell cycle phase distribution, suggesting a driver role in cancer progression.

A Sox2-Sox9 signalling axis maintains human breast luminal progenitor and breast cancer stem cells.

Increased cancer stem cell content during development of resistance to tamoxifen in breast cancer is driven by multiple signals, including Sox2-dependent activation of Wnt signalling. Here, we show that Sox2 increases and estrogen reduces the expression of the transcription factor Sox9. Gain and loss of function assays indicate that Sox9 is implicated in the maintenance of human breast luminal progenitor cells. CRISPR/Cas knockout of Sox9 reduces growth of tamoxifen-resistant breast tumours in vivo. Mechanistically, Sox9 acts downstream of Sox2 to control luminal progenitor cell content and is required for expression of the cancer stem cell marker ALDH1A3 and Wnt signalling activity. Sox9 is elevated in breast cancer patients after endocrine therapy failure. This new regulatory axis highlights the relevance of SOX family transcription factors as potential therapeutic targets in breast cancer.

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1.

Aberrant transforming growth factor-ß (TGF-ß) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-ß signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-ß signaling activity and that stromal cell-conditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-ß-induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment.

Distinct breast cancer stem/progenitor cell populations require either HIF1α or loss of PHD3 to expand under hypoxic conditions.

The heterogeneous nature of breast cancer is a result of intrinsic tumor complexity and also of the tumor microenvironment, which is known to be hypoxic. We found that hypoxia expands different breast stem/progenitor cell populations (cells with increased aldehyde dehydrogenase activity (Aldefluor+), high mammosphere formation capacity and CD44+CD24-/low cells) both in primary normal epithelial and tumor cells. The presence of the estrogen receptor (ER) limits hypoxia-dependent CD44+CD24-/low cell expansion.We further show that the hypoxia-driven cancer stem-like cell enrichment results from a dedifferentiation process. The enhanced mammosphere formation and Aldefluor+ cell content observed in breast cancer cells relies on hypoxia-inducible factor 1α (HIF1α). In contrast, the CD44+CD24-/low population expansion is HIF1α independent and requires prolyl hydroxylase 3 (PHD3) downregulation, which mimics hypoxic conditions, leading to reduced CD24 expression through activation of NFkB signaling. These studies show that hypoxic conditions expand CSC populations through distinct molecular mechanisms. Thus, potential therapies that combine current treatments for breast cancer with drugs that target CSC should take into account the heterogeneity of the CSC subpopulations.

FACS Sorting Mammary Stem Cells.

Fluorescent-activated cell sorting (FACS) represents one of the key techniques that have been used to isolate and characterize stem cells, including cells from the mammary gland. A combination of approaches, including recognition of cell surface antigens and different cellular activities, has facilitated the identification of stem cells from the healthy mammary gland and from breast tumors. In this chapter we describe the protocol to use FACS to separate breast cancer stem cells, but most of the general principles discussed could be applied to sort other types of cells.

Dickkopf-3 alters the morphological response to retinoic acid during neuronal differentiation of human embryonal carcinoma cells.

Dickkopf-3 (Dkk-3) and Dkkl-1 (Soggy) are secreted proteins of poorly understood function that are highly expressed in subsets of neurons in the brain. To explore their potential roles during neuronal development, we examined their expression in Ntera-2 (NT2) human embryonal carcinoma cells, which differentiate into neurons upon treatment with retinoic acid (RA). RA treatment increased the mRNA and protein levels of Dkk-3 but not of Dkkl-1. Ectopic expression of both Dkk-3 and Dkkl-1 induced apoptosis in NT2 cells. Gene silencing of Dkk-3 did not affect NT2 cell growth or differentiation but altered their response to RA in suspension cultures. RA treatment of NT2 cells cultured in suspension resulted in morphological changes that led to cell attachment and flattening out of cell aggregates. Although there were no significant differences in the expression levels of cell adhesion molecules in control and Dkk-3-silenced cells, this morphological response was not observed in Dkk-3-silenced cells. These findings suggest that Dkk-3 plays a role in the regulation of cell interactions during RA-induced neuronal differentiation.

Sox2 promotes tamoxifen resistance in breast cancer cells.

Development of resistance to therapy continues to be a serious clinical problem in breast cancer management. Cancer stem/progenitor cells have been shown to play roles in resistance to chemo­ and radiotherapy. Here, we examined their role in the development of resistance to the oestrogen receptor antagonist tamoxifen. Tamoxifen­resistant cells were enriched for stem/progenitors and expressed high levels of the stem cell marker Sox2. Silencing of the SOX2 gene reduced the size of the stem/progenitor cell population and restored sensitivity to tamoxifen. Conversely, ectopic expression of Sox2 reduced tamoxifen sensitivity in vitro and in vivo. Gene expression profiling revealed activation of the Wnt signalling pathway in Sox2­expressing cells, and inhibition of Wnt signalling sensitized resistant cells to tamoxifen. Examination of patient tumours indicated that Sox2 levels are higher in patients after endocrine therapy failure, and also in the primary tumours of these patients, compared to those of responders. Together, these results suggest that development of tamoxifen resistance is driven by Sox2­dependent activation of Wnt signalling in cancer stem/progenitor cells.

Sphingosine-1-phosphate stimulates aldosterone secretion through a mechanism involving the PI3K/PKB and MEK/ERK 1/2 pathways.

We reported recently that sphingosine-1-phosphate (S1P) is a novel regulator of aldosterone secretion in zona glomerulosa cells of adrenal glands and that phospholipase D (PLD) is implicated in this process. We now show that S1P causes the phosphorylation of protein kinase B (PKB) and extracellularly regulated kinases 1/2 (ERK 1/2), which is an indication of their activation, in these cells. These effects are probably mediated through the interaction of S1P with the Gi protein-coupled receptors S1P1/3, as pretreatment with pertussis toxin or with the S1P1/3 antagonist VPC 23019 completely abolished the phosphorylation of these kinases. Inhibitors of phosphatidylinositol 3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK) blocked S1P-stimulated aldosterone secretion. This inhibition was only partial when the cells were incubated independently with inhibitors of each pathway. However, aldosterone output was completely blocked when the cells were pretreated with LY 294002 and PD 98059 simultaneously. These inhibitors also blocked PLD activation, which indicates that this enzyme is downstream of PI3K and MEK in this system. We propose a working model for S1P in which stimulation of the PI3K/PKB and MEK/ERK pathways leads to the stimulation of PLD and aldosterone secretion.