Essential role for centromeric factors following p53 loss and oncogenic transformation.

In mammals, centromere definition involves the histone variant CENP-A (centromere protein A), deposited by its chaperone, HJURP (Holliday junction recognition protein). Alterations in this process impair chromosome segregation and genome stability, which are also compromised by p53 inactivation in cancer. Here we found that CENP-A and HJURP are transcriptionally up-regulated in p53-null human tumors. Using an established mouse embryonic fibroblast (MEF) model combining p53 inactivation with E1A or HRas-V12 oncogene expression, we reproduced a similar up-regulation of HJURP and CENP-A. We delineate functional CDE/CHR motifs within the Hjurp and Cenpa promoters and demonstrate their roles in p53-mediated repression. To assess the importance of HJURP up-regulation in transformed murine and human cells, we used a CRISPR/Cas9 approach. Remarkably, depletion of HJURP leads to distinct outcomes depending on their p53 status. Functional p53 elicits a cell cycle arrest response, whereas, in p53-null transformed cells, the absence of arrest enables the loss of HJURP to induce severe aneuploidy and, ultimately, apoptotic cell death. We thus tested the impact of HJURP depletion in pre-established allograft tumors in mice and revealed a major block of tumor progression in vivo. We discuss a model in which an "epigenetic addiction" to the HJURP chaperone represents an Achilles' heel in p53-deficient transformed cells.

Regulation of transgenes in three-dimensional cultures of primary mouse mammary cells demonstrates oncogene dependence and identifies cells that survive deinduction.

The advent of targeted therapies for cancer has provoked interest in experimental models for the systematic study of oncogene dependence. To that end, we developed a three-dimensional (3D) culture system to analyze the responses of primary mouse mammary epithelial cells to the induction and deinduction of oncogenes. Mammary cells derived from normal virgin mice, or from tritransgenic mice (TetO-MYC;TetO-Kras(G12D);MMTV-rtTA) in which MYC and mutant Kras can be regulated by doxycycline, develop from single cells into polarized acini. Lumen formation occurs without apparent apoptosis, and the hollow spheres of cells enlarge by division, with metaphase plates oriented perpendicularly to the apical surface. When MYC and Kras(G12D) are induced, the acini enlarge and form solid, depolarized spheres. Upon deinduction of MYC and Kras(G12D) the solid structures regress, leaving a repolarized monolayer of viable cells. These cells display a phenotype consistent with progenitors of mammary epithelium: They exclude Hoechst dye 33342, and reform acini in 3D cultures and repopulate mammary fat pads more efficiently than cells harvested from uninduced acini. Moreover, cells in the surviving spheres retain the ability to respond to reinduction and thus may represent the type of cells that give rise to recurrent tumors.

Defining the ATM-mediated barrier to tumorigenesis in somatic mammary cells following ErbB2 activation.

p53, apoptosis, and senescence are frequently activated in preneoplastic lesions and are barriers to progression to malignancy. These barriers have been suggested to result from an ATM-mediated DNA damage response (DDR), which may follow oncogene-induced hyperproliferation and ensuing DNA replication stress. To elucidate the currently untested role of DDR in breast cancer initiation, we examined the effect of oncogene expression in several murine models of breast cancer. We did not observe a detectable DDR in early hyperplastic lesions arising in transgenic mice expressing several different oncogenes. However, DDR signaling was strongly induced in preneoplastic lesions arising from individual mammary cells transduced in vivo by retroviruses expressing either PyMT or ErbB2. Thus, activation of an oncogene after normal tissue development causes a DDR. Furthermore, in this somatic ErbB2 tumor model, ATM, and thus DDR, is required for p53 stabilization, apoptosis, and senescence. In palpable tumors in this model, p53 stabilization and apoptosis are lost, but unexpectedly senescence remains in many tumor cells. Thus, this murine model fully recapitulates early DDR signaling; the eventual suppression of its endpoints in tumorigenesis provides compelling evidence that ErbB2-induced aberrant mammary cell proliferation leads to an ATM-mediated DDR that activates apoptosis and senescence, and at least the former must be overcome to progress to malignancy. This in vivo study also uncovers an unexpected effect of ErbB2 activation previously known for its prosurvival roles, and suggests that protection of the ATM-mediated DDR-p53 signaling pathway may be important in breast cancer prevention.

The role of the antigen processing machinery in the regulation and trafficking of intracellular -Toll-like receptor molecules.

Intracellular Toll-like receptors (TLRs) are key components of the innate immune system. Their expression in antigen-presenting cells (APCs), and in particular dendritic cells (DCs), makes them critical in the induction of the adaptive immune response. In DCs, they interact with the chaperone UNC93B1 that mediates their trafficking from the endoplasmic reticulum (ER) to endosomes where they are cleaved by proteases and activated. All these different steps are also shared by major histocompatibility complex class-II (MHCII) molecules. Here, we will discuss the tight relationship intracellular TLRs have with the antigen processing machinery in APCs for their trafficking and activation.

FLT3L-dependent dendritic cells control tumor immunity by modulating Treg and NK cell homeostasis.

FLT3-L-dependent classical dendritic cells (cDCs) recruit anti-tumor and tumor-protecting lymphocytes. We evaluate cancer growth in mice with low, normal, or high levels of cDCs. Paradoxically, both low or high numbers of cDCs improve survival in mice with melanoma. In low cDC context, tumors are restrained by the adaptive immune system through influx of effector T cells and depletion of Tregs and NK cells. High cDC numbers favor the innate anti-tumor response, with massive recruitment of activated NK cells, despite high Treg infiltration. Anti CTLA-4 but not anti PD-1 therapy synergizes with FLT3-L therapy in the cDCHi but not in the cDCLo context. A combination of cDC boost and Treg depletion dramatically improves survival of tumor-bearing mice. Transcriptomic data confirm the paradoxical effect of cDC levels on survival in several human tumor types. cDCHi-TregLo state in such patients predicts best survival. Modulating cDC numbers via FLT3 signaling may have therapeutic potential in human cancer.

The role of endoplasmic reticulum stress in the MHC class I antigen presentation pathway of dendritic cells.

Dendritic cells (DCs) have the unique capacity to link innate to adaptive immunity. While most cells that express major histocompatibility (MHC) molecules are able to present antigens to activated T cells, DCs possess the means for presenting antigens to naïve T cells, and, as such, are able to instruct T cells to initiate immune response. There are two cascades of events necessary for DCs to start their instructive function. First, DCs enzymatically process proteins to make T cells recognize an antigen as unique peptide-MHC complexes. Second, DCs provide secretory cytokines and co-stimulatory functions for T cells to respond to this antigen. Thus, the compartments for protein degradation and for protein synthesis are central to DC function. The endoplasmic reticulum (ER), a vast network of membranes and vesicles, connects these compartments and helps modulate DC-specific performance, such as antigen capture and presentation. However, while the health of ER appears relevant for DC function, the intersection between ER stress and antigen presentation remains to be explored.

Oncogene cooperation in tumor maintenance and tumor recurrence in mouse mammary tumors induced by Myc and mutant Kras.

Most, if not all, cancers are composed of cells in which more than one gene has a cancer-promoting mutation. Although recent evidence has shown the benefits of therapies targeting a single mutant protein, little attention has been given to situations in which experimental tumors are induced by multiple cooperating oncogenes. Using combinations of doxycycline-inducible and constitutive Myc and mutant Kras transgenes expressed in mouse mammary glands, we show that tumors induced by the cooperative actions of two oncogenes remain dependent on the activity of a single oncogene. Deinduction of either oncogene individually, or both oncogenes simultaneously, led to partial or complete tumor regression. Prolonged remission followed deinduction of Kras(G12D) in the context of continued Myc expression, deinduction of a MYC transgene with continued expression of mutant Kras produced modest effects on life extension, whereas simultaneous deinduction of both MYC and Kras(G12D) transgenes further improved survival. Disease relapse after deinduction of both oncogenes was associated with reactivation of both oncogenic transgenes in all recurrent tumors, often in conjunction with secondary somatic mutations in the tetracycline transactivator transgene, MMTV-rtTA, rendering gene expression doxycycline-independent. These results demonstrate that tumor viability is maintained by each gene in a combination of oncogenes and that targeted approaches will also benefit from combination therapies.

CENP-A overexpression promotes distinct fates in human cells, depending on p53 status.

Tumour evolution is driven by both genetic and epigenetic changes. CENP-A, the centromeric histone H3 variant, is an epigenetic mark that directly perturbs genetic stability and chromatin when overexpressed. Although CENP-A overexpression is a common feature of many cancers, how this impacts cell fate and response to therapy remains unclear. Here, we established a tunable system of inducible and reversible CENP-A overexpression combined with a switch in p53 status in human cell lines. Through clonogenic survival assays, single-cell RNA-sequencing and cell trajectory analysis, we uncover the tumour suppressor p53 as a key determinant of how CENP-A impacts cell state, cell identity and therapeutic response. If p53 is functional, CENP-A overexpression promotes senescence and radiosensitivity. Surprisingly, when we inactivate p53, CENP-A overexpression instead promotes epithelial-mesenchymal transition, an essential process in mammalian development but also a precursor for tumour cell invasion and metastasis. Thus, we uncover an unanticipated function of CENP-A overexpression to promote cell fate reprogramming, with important implications for development and tumour evolution.

Glycolipid-dependent and lectin-driven transcytosis in mouse enterocytes.

Glycoproteins and glycolipids at the plasma membrane contribute to a range of functions from growth factor signaling to cell adhesion and migration. Glycoconjugates undergo endocytic trafficking. According to the glycolipid-lectin (GL-Lect) hypothesis, the construction of tubular endocytic pits is driven in a glycosphingolipid-dependent manner by sugar-binding proteins of the galectin family. Here, we provide evidence for a function of the GL-Lect mechanism in transcytosis across enterocytes in the mouse intestine. We show that galectin-3 (Gal3) and its newly identified binding partner lactotransferrin are transported in a glycosphingolipid-dependent manner from the apical to the basolateral membrane. Transcytosis of lactotransferrin is perturbed in Gal3 knockout mice and can be rescued by exogenous Gal3. Inside enterocytes, Gal3 is localized to hallmark structures of the GL-Lect mechanism, termed clathrin-independent carriers. These data pioneer the existence of GL-Lect endocytosis in vivo and strongly suggest that polarized trafficking across the intestinal barrier relies on this mechanism.

Differential interleukin-6/Stat3 signaling as a function of cellular context mediates Ras-induced transformation.

INTRODUCTION: Tyrosine phosphorylated signal transducer and activator of transcription 3 (pStat3) is present in numerous cancers and is required for mediating tumorigenesis. Autocrine and paracrine interleukin (IL)-6 signaling is the principal mechanism by which Stat3 is persistently phosphorylated in epithelial tumors including breast, lung, colon and gastric cancer. The Ras oncogene mediates cellular transformation without evidence of pStat3 in cultured cells. Recently, however non-tyrosine phosphorylated Stat3 was shown to have a transcriptional activating function, a role in mitochondrial function and to mediate cell migration.. Here we examined the role of Stat3 in Ras mediated transformation. METHODS: Ha-rasV12 transformed mammary epithelial cells (MCF10A) cells were transduced with a Stat3shRNA, IL-6shRNA and/or treated with inhibitors of Janus kinases (JAKs) to examine the role of the IL-6 signaling pathway in Ras mediated invasion and tumorigenesis. RESULTS: Cellular migration, invasion, anchorage independent growth and tumorigenesis were largely abrogated in the Stat3-reduced cells compared to control cells. Analysis of MCF10A-Ras tumors revealed high levels of pStat3 and interleukin-6. Tumors derived from transgenic MMTV-K-Ras mice were also found to express pStat3 and IL-6. MCF10A-Ras cells, when grown in a three-dimensional Matrigel culture system revealed the appearance of the junctional protein E-Cadherin as a consequence of reducing Stat3 levels or inhibiting Stat3 activity. Decreasing IL-6 levels in the MCF10A-Ras cells abrogated tumorigenesis and reduced cell migration. By isolating Ras-expressing primary tumors and serially passaging these cells in two-dimensional culture led to a decrease in IL-6 and pStat3 levels with the reappearance of E-Cadherin. CONCLUSIONS: The cellular and environmental context can lead to differential IL-6/pStat3 signaling and a dependency on this cytokine and transcription factor for migration, invasion and tumorigenesis.

Shaping Chromatin in the Nucleus: The Bricks and the Architects.

Chromatin organization in the nucleus provides a vast repertoire of information in addition to that encoded genetically. Understanding how this organization impacts genome stability and influences cell fate and tumorigenesis is an area of rapid progress. Considering the nucleosome, the fundamental unit of chromatin structure, the study of histone variants (the bricks) and their selective loading by histone chaperones (the architects) is particularly informative. Here, we report recent advances in understanding how relationships between histone variants and their chaperones contribute to tumorigenesis using cell lines and Xenopus development as model systems. In addition to their role in histone deposition, we also document interactions between histone chaperones and other chromatin factors that govern higher-order structure and control DNA metabolism. We highlight how a fine-tuned assembly line of bricks (H3.3 and CENP-A) and architects (HIRA, HJURP, and DAXX) is key in adaptation to developmental and pathological changes. An example of this conceptual advance is the exquisite sensitivity displayed by p53-null tumor cells to modulation of HJURP, the histone chaperone for CENP-A (CenH3 variant). We discuss how these findings open avenues for novel therapeutic paradigms in cancer care.

Estrogen receptor positivity in mammary tumors of Wnt-1 transgenic mice is influenced by collaborating oncogenic mutations.

The majority (75%) of human breast cancers express estrogen receptor (ER). Although ER-positive tumors usually respond to antiestrogen therapies, 30% of them do not. It is not known what controls the ER status of breast cancers or their responsiveness to antihormone interventions. In this report, we document that transgenic (TG) expression of Wnt-1 in mice induces ER-positive tumors. Loss of Pten or gain of Ras mutations during the evolution of tumors in Wnt-1 TG mice has no effect on the expression of ER, but overexpression of Neu or loss of p53 leads to ER-negative tumors. Thus, our results provide compelling evidence that expression of ER in breast cancer may be influenced by specific genetic changes that promote cancer progression. These findings constitute a first step to explore the molecular mechanisms leading to ER-positive or ER-negative mammary tumors. In addition, we find that ER-positive tumors arising in Wnt-1 TG mice are refractory to both ovariectomy and the ER antagonist tamoxifen, but lose ER expression with tamoxifen, suggesting that antiestrogen selects for ER-negative tumor cells and that the ER-positive cell fraction is dispensable for growth of these tumors. This is a first report of a mouse model of antiestrogen-resistant ER-positive breast cancers, and could provide a powerful tool to study the molecular mechanisms that control antiestrogen resistance.

Analysis of PTEN mutation in non-familial pheochromocytoma.

PTEN, a tumor suppressor gene, is frequently mutated in a variety of human tumors. In mice, monoallelic inactivation of this gene predisposes animals to neoplasia of multiple organs. Interestingly, Pten heterozygous mice develop bilateral hyperplasia of the adrenal medulla. In this report we demonstrate that these neoplasms are hormonally active pheochromocytomas that secrete increased amounts of bioactive catecholamines: norepinephrine and epinephrine. To test a possibility that PTEN might be one of the genes responsible for human sporadic pheochromocytoma, we performed mutation analysis of DNA obtained from tumors of 29 patients. However, direct sequencing of all nine exons of the PTEN gene, including the splice junctions, revealed no mutations. Examination of protein expression by immunohistochemistry using 8 normal adrenals and 11 sporadic pheochromocytomas showed no decrease in the PTEN protein expression in the tumor tissue, but upregulation of insulin-like growth factor II, a peptide implicated in growth of adrenal tissue, was observed in four cases (36%).

Wnt-1 is dominant over neu in specifying mammary tumor expression profiles.

Wnt-1 and Neu collaborate to induce mammary tumors in bitransgenic mice carrying both MMTV-Wnt-1 and MMTV-Neu. In this report, gene expression profiles were determined for tumors from these bitransgenic mice, and compared with expression profiles of tumors from mice singly transgenic for MMTV-Wnt-1 or MMTV-Neu. While very different from tumors arising in MMTV-Neu transgenic mice, tumors from these bitransgenic mice were found not to have identifiable differences from tumors from MMTV-Wnt-1 transgenic mice, using clustering and multidimensional scaling analyses (unsupervised and supervised), One-way Analysis of Variance (ANOVA), and two sample t test (the later two of which were combined with false discovery rate computation). These observations suggest that Wnt-1 is dominant over Neu in specifying mammary tumor expression profiles.

Tissue Dissociation for Metastasis Studies.

The main requirement for most metastasis-related applications is the conversion of solid tissue into a single-cell suspension. In theory, this suspension represents the diversity of cells present in the tissue, whether malignant or benign. We have found that cell viability, as measured by trypan blue staining or fluorescence-activated cell sorting (FACS), is critical for evaluating the success of the tissue-dissociation procedure. The recommended goal is at least 70% cell viability.

Social networking in tumor cell communities is associated with increased aggressiveness.

Extracellular vesicles (EVs) are lipid-bilayer-enclosed vesicles that contain proteins, lipids and nucleic acids. EVs produced by cells from healthy tissues circulate in the blood and body fluids, and can be taken up by unrelated cells. As they have the capacity to transfer cargo proteins, lipids and nucleic acids (mostly mRNAs and miRNAs) between different cells in the body, EVs are emerging as mediators of intercellular communication that could modulate cell behavior, tissue homeostasis and regulation of physiological functions. EV-mediated cell-cell communications are also proposed to play a role in disease, for example, cancer, where they could contribute to transfer of traits required for tumor progression and metastasis. However, direct evidence for EV-mediated mRNA transfer to individual cells and for its biological consequences in vivo has been missing until recently. Recent studies have reported elegant experiments using genetic tracing with the Cre recombinase system and intravital imaging that visualize and quantify functional transfer of mRNA mediated by EVs in the context of cancer and metastasis.

Murine Stem Cell-Based Retrovirus Production for Marking Primary Mouse Mammary Cells for Metastasis Studies.

Since the introduction of retroviral vector technology, permanent genetic marking of cells has considerably contributed to the understanding of different physiological and disease processes in vivo. Recent marking strategies aim to elucidate the contribution of cells on the clonal level, and the advent of fluorescent proteins has opened new avenues for the in vivo analysis of gene-marked cells. Gene-modified cells are easily identifiable (e.g., via the introduced fluorescent protein) within whole organ structures, allowing one to measure the contribution of transduced cells to malignant outgrowth. In our laboratory, we use the tetracycline-inducible system to study oncogene cooperation in metastatic progression. We use bicistronic retroviruses expressing the tetracycline transactivator (tTA) and the candidate gene (MIT-gene) or the tTA alone (MIT-Rx) to infect primary mammary cells from mice harboring tetracycline-inducible transgenes. This allows for constitutive expression of the candidate gene and tTA-dependent expression of the inducible oncogene. We also use MIG-based vectors, which allow for constitutive expression of the candidate gene and a green fluorescent protein. Here we describe how to produce retroviral particles carrying both MIT- and MIG-based vectors. Because of the fragility of the retroviral envelope, we do not attempt to concentrate the virus, and we directly use packaging cell media to infect primary epithelial cells (either normal or tumor). Infected cells can be transplanted into recipient mice to investigate metastatic colonization.

Enrichment of Mammary Basal and Luminal Cells for Cell-of-Origin Metastasis Studies.

The mammary gland is an important model system in metastasis research. Mammary epithelial stem cells are of particular interest because of their capacity for regeneration and their role in cancer initiation. This protocol describes how to enrich for mammary basal and luminal epithelial cells using fluorescence-activated cell sorting (FACS).

Methods to Study Metastasis in Genetically Modified Mice.

Metastasis is often modeled by xenotransplantation of cell lines in immunodeficient mice. A wealth of information about tumor cell behavior in the new environment is obtained from these efforts. Yet by design, this approach is "tumor-centric," as it focuses on cell-autonomous determinants of human tumor dissemination in mouse tissues, in effect using the animal body as a sophisticated "Petri dish" providing nutrients and support for tumor growth. Transgenic or gene knockout mouse models of cancer allow the study of tumor spread as a systemic disease and offer a complimentary approach for studying the natural history of cancer. This introduction is aimed at describing the overall methodological approach to studying metastasis in genetically modified mice, with a particular focus on using animals with regulated expression of potent human oncogenes in the breast.

Methods to study primary tumor cells and residual tumor cells in mouse models of oncogene dependence.

The studies of oncogene dependence are aimed to understand an unfortunate and puzzling aspect of targeted anticancer treatments-their progression to drug resistance. Drug resistance develops from a pool of cells that survive the original treatment, called minimal residual disease. Mouse models based on tetracycline-dependent expression of transgenic oncogenes are used to imitate targeted oncogene blockade and to reproduce minimal residual disease in humans. Here we describe a novel method for generating oncogene-dependent mammary tumors using somatic transfer of transactivator-containing retroviruses into transgenic mice with tetracycline-dependent oncogenes and a method for measuring continuous mitotic activity in epithelial cells in real time.