C/EBPδ links IL-6 and HIF-1 signaling to promote breast cancer stem cell-associated phenotypes.

To improve cancer patient outcome significantly, we must understand the mechanisms regulating stem-like cancer cells, which have been implicated as a cause of metastasis and treatment resistance. The transcription factor C/EBPδ can exhibit pro- and anti-tumorigenic activities, but the mechanisms underlying the complexity of its functions are poorly understood. Here we identify a role for breast cancer cell intrinsic C/EBPδ in promoting phenotypes that have been associated with cancer stem cells (CSCs). While C/EBPδ expression is not abundant in most metastatic breast cancers, our data support a pro-tumorigenic role of C/EBPδ when expressed in subsets of tumor cells and/or through transient activation by the tumor microenvironment or loss of substrate adhesion. Using genetic mouse models and human breast cancer cell lines, we show that deletion or depletion of C/EBPδ reduced expression of stem cell factors and stemnness markers, sphere formation and self-renewal, along with growth of tumors and established experimental metastases in vivo. C/EBPδ is also known as a mediator of the innate immune response, which is enhanced by hypoxia and interleukin-6 (IL-6) signaling, two conditions that also play important roles in cancer progression. Our mechanistic data reveal C/EBPδ as a link that engages two positive feedback loops, in part by directly targeting the IL-6 receptor (IL6RA) gene, and, thus, amplifying IL-6 and HIF-1 signaling. This study provides a molecular mechanism for the synergism of tumor microenvironmental conditions in cancer progression with potential implications for the targeting of CSCs.

Organoid Modeling of the Tumor Immune Microenvironment.

In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.

Early synergistic interactions between the HPV16­E7 oncoprotein and 17ß-oestradiol for repressing the expression of Granzyme B in a cervical cancer model.

Although high-risk human papillomavirus (HR­HPV) infection has a prominent role in the aetiology of cervical cancer (CC), sex steroid hormones may also be involved in this process; however, the cooperation between oestrogen and HR­HPV in the early stages of cervical carcinogenesis is poorly understood. Since 17ß-oestradiol (E2) and the HPV type 16­E7 oncoprotein induce CC in transgenic mice, a microarray analysis was performed in the present study to generate global gene expression profiles from 2­month­old FVB (non­transgenic) and K14E7 (transgenic) mice who were left untreated or were treated for 1 month with E2. Upregulation of cancer-related genes that have not been previously reported in the context of CC, including glycerophosphodiester phosphodiesterase domain containing 3, interleukin 1 receptor type II, natriuretic peptide type C, MGAT4 family member C, lecithin-retinol acyltransferase (phosphatidylcholine-retinol-O-acyltransferase) and glucoside xylosyltransferase 2, was observed. Notably, upregulation of the serine (or cysteine) peptidase inhibitor clade B member 9 gene and downregulation of the Granzyme gene family were observed; the repression of the Granzyme B pathway may be a novel mechanism of immune evasion by cancer cells. The present results provide the basis for further studies on early biomarkers of CC risk and synergistic interactions between HR­HPV and oestrogen.

The PDZ-Binding Motif of HPV16-E6 Oncoprotein Modulates the Keratinization and Stemness Transcriptional Profile In Vivo.

OBJECTIVE: The aim of this work was to compare the early gene expression profiles in the skin of HPV16-E6 transgenic mice regulated by the E6 PDZ-binding motif. MATERIALS AND METHODS: The global transcriptional profiles in dorsal skin biopsies from K14E6 and K14E6Δ146-151 transgenic mice were compared using microarrays. Relevant genes obtained from the most differentially expressed processes were further examined by RT-qPCR, in situ RT-PCR, Western blot, or immunofluorescence. RESULTS: The transcriptomic landscape of K14E6 versus K14E6Δ146-151 shows that the most affected expression profiles were those related to keratinocyte differentiation, stem cell maintenance, and keratinization. Additionally, downregulation of epidermal stemness markers such as K15 and CD34, as well as the upregulation of cytokeratin 6b, appeared to be dependent on the E6 PDZ-binding motif. Finally, wound healing, a physiological process linked to stemness, is impaired in the K14E6 mice compared to K14E6Δ146-151. CONCLUSION: The E6 PDZ-binding motif appears to affect stemness and keratinization during early stages of skin carcinogenesis. As E6 plays a significant role in HPV-induced skin carcinogenesis, the K14E6 versus K14E6Δ146-151 transcriptional profile provides a source of valuable data to uncover novel E6 functions in the skin.

Runx Genes in Breast Cancer and the Mammary Lineage.

A full understanding of RUNX gene function in different epithelial lineages has been thwarted by the lethal phenotypes observed when constitutively knocking out these mammalian genes. However temporal expression of the Runx genes throughout the different phases of mammary gland development is indicative of a functional role in this tissue. A few studies have emerged describing how these genes impact on the fate of mammary epithelial cells by regulating lineage differentiation and stem/progenitor cell potential, with implications for the transformed state. The importance of the RUNX/CBFß core factor binding complex in breast cancer has very recently been highlighted with both RUNX1 and CBFß appearing in a comprehensive gene list of predicted breast cancer driver mutations. Nonetheless, the evidence to date shows that the RUNX genes can have dualistic outputs with respect to promoting or constraining breast cancer phenotypes, and that this may be aligned to individual subtypes of the clinical disease. We take this opportunity to review the current literature on RUNX and CBFß in the normal and neoplastic mammary lineage while appreciating that this is likely to be the tip of the iceberg in our knowledge.

Gene expression profile regulated by the HPV16 E7 oncoprotein and estradiol in cervical tissue.

The HPV16 E7 oncoprotein and 17ß-estradiol are important factors for the induction of premalignant lesions and cervical cancer. The study of these factors is crucial for a better understanding of cervical tumorigenesis. Here, we assessed the global gene expression profiles induced by the HPV16 E7 oncoprotein and/or 17ß-estradiol in cervical tissue of FvB and K14E7 transgenic mice. We found that the most dramatic changes in gene expression occurred in K14E7 and FvB groups treated with 17ß-estradiol. A large number of differentially expressed genes involved in the immune response were observed in 17ß-estradiol treated groups. The E7 oncoprotein mainly affected the expression of genes involved in cellular metabolism. Our microarray data also identified differentially expressed genes that have not previously been reported in cervical cancer. The identification of genes regulated by E7 and 17ß-estradiol, provides the basis for further studies on their role in cervical carcinogenesis.

Cytoskeletal protein filamin A is a nucleolar protein that suppresses ribosomal RNA gene transcription.

Filamin A (FLNA) is an actin-binding protein with a well-established role in the cytoskeleton, where it determines cell shape and locomotion by cross-linking actin filaments. Mutations in FLNA are associated with a wide range of genetic disorders. Here we demonstrate a unique role for FLNA as a nucleolar protein that associates with the RNA polymerase I (Pol I) transcription machinery to suppress rRNA gene transcription. We show that depletion of FLNA by siRNAs increased rRNA expression, rDNA promoter activity and cell proliferation. Immunodepletion of FLNA from nuclear extracts resulted in a decrease in rDNA promoter-driven transcription in vitro. FLNA coimmunoprecipitated with the Pol I components actin, TIF-IA, and RPA40, and their occupancy of the rDNA promoter was increased in the absence of FLNA in vivo. The FLNA actin-binding domain is essential for the suppression of rRNA expression and for inhibiting recruitment of the Pol I machinery to the rDNA promoter. These findings reveal an additional role for FLNA as a regulator of rRNA gene expression and have important implications for our understanding of the role of FLNA in human disease.

Metastatic breast cancer cells inhibit osteoblast differentiation through the Runx2/CBFß-dependent expression of the Wnt antagonist, sclerostin.

INTRODUCTION: Breast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction by stimulating osteoclasts to resorb bone and by preventing osteoblasts from producing new bone. The Runt-related transcription factor 2, Runx2, is an important determinant of bone metastasis in breast cancer. Runx2 is known to mediate activation of osteoclast activity and inhibition of osteoblast differentiation by metastatic breast cancer cells. However, while Runx2-regulated genes that mediate osteoclast activation have been identified, how Runx2 determines inhibition of osteoblasts is unknown. METHODS: The aim of this study was to determine how Runx2 mediates the ability of metastatic breast cancer cells to modulate the activity of bone cells. We have previously demonstrated that Runx2 requires the co-activator core binding factor beta (CBFß) to regulate gene expression in breast cancer cells. We, therefore, performed independent microarray analyses to identify target genes whose expression is dependent upon both Runx2 and CBFß. Common target genes, with a role in modulating bone-cell function, were confirmed using a combination of siRNA, quantitative reverse transcriptase PCR (qRT-PCR), ELISA, promoter reporter analysis, Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The function of Runx2/CBFß-regulated genes in mediating the ability of MDA-MB-231 to inhibit osteoblast differentiation was subsequently established in primary bone marrow stromal cell cultures and MC-3T3 osteoblast cells. RESULTS: We show that Runx2/CBFß mediates inhibition of osteoblast differentiation by MDA-MB-231 cells through induction of the Wnt signaling antagonist, sclerostin. We demonstrate that MDA-MB-231 cells secrete sclerostin and that sclerostin-expression is critically dependent on both Runx2 and CBFß. We also identified the osteoclast activators IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as new target genes of Runx2/CBFß in metastatic breast cancer cells. CONCLUSIONS: This study demonstrates that Runx2 and CBFß are required for the expression of genes that mediate the ability of metastatic breast cancer cells to directly modulate both osteoclast and osteoblast function. We also show that Runx2-dependent inhibition of osteoblast differentiation by breast cancer cells is mediated through the Wnt antagonist, sclerostin.

The Runx transcriptional co-activator, CBFbeta, is essential for invasion of breast cancer cells.

BACKGROUND: The transcription factor Runx2 has an established role in cancers that metastasize to bone. In metastatic breast cancer cells Runx2 is overexpressed and contributes to the invasive capacity of the cells by regulating the expression of several invasion genes. CBFbeta is a transcriptional co-activator that is recruited to promoters by Runx transcription factors and there is considerable evidence that CBFbeta is essential for the function of Runx factors. However, overexpression of Runx1 can partially rescue the lethal phenotype in CBFbeta-deficient mice, indicating that increased levels of Runx factors can, in some situations, overcome the requirement for CBFbeta. Since Runx2 is overexpressed in metastatic breast cancer cells, and there are no reports of CBFbeta expression in breast cells, we sought to determine whether Runx2 function in these cells was dependent on CBFbeta. Such an interaction might represent a viable target for therapeutic intervention to inhibit bone metastasis. RESULTS: We show that CBFbeta is expressed in the metastatic breast cancer cells, MDA-MB-231, and that it associates with Runx2. Matrigel invasion assays and RNA interference were used to demonstrate that CBFbeta contributes to the invasive capacity of these cells. Subsequent analysis of Runx2 target genes in MDA-MB-231 cells revealed that CBFbeta is essential for the expression of Osteopontin, Matrixmetalloproteinase-13, Matrixmetalloproteinase-9, and Osteocalcin but not for Galectin-3. Chromatin immunoprecipitation analysis showed that CBFbeta is recruited to both the Osteopontin and the Galectin-3 promoters. CONCLUSIONS: CBFbeta is expressed in metastatic breast cancer cells and is essential for cell invasion. CBFbeta is required for expression of several Runx2-target genes known to be involved in cell invasion. However, whilst CBFbeta is essential for invasion, not all Runx2-target genes require CBFbeta. We conclude that CBFbeta is required for a subset of Runx2-target genes that are sufficient to maintain the invasive phenotype of the cells. These findings suggest that the interaction between Runx2 and CBFbeta might represent a viable target for therapeutic intervention to inhibit bone metastasis.