Felicite K. Noubissi

CRD-BP mediates stabilization of βTrCP1 and c- myc mRNA in response to β-catenin signalling

Although constitutive activation of β-catenin/Tcf signalling is implicated in the development of human cancers, the mechanisms by which the β-catenin/Tcf pathway promotes tumorigenesis are incompletely understood. Messenger RNA turnover has a major function in regulating gene expression and is responsive to developmental and environmental signals. mRNA decay rates are dictated by cis-acting elements within the mRNA and by trans-acting factors, such as RNA-binding proteins (reviewed in refs 2, 3). Here we show that β-catenin stabilizes the mRNA encoding the F-box protein βTrCP1, and identify the RNA-binding protein CRD-BP (coding region determinant-binding protein) as a previously unknown target of β-catenin/Tcf transcription factor. CRD-BP binds to the coding region of βTrCP1 mRNA. Overexpression of CRD-BP stabilizes βTrCP1 mRNA and elevates βTrCP1 levels (both in cells and in vivo), resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)βTrCP E3 ubiquitin ligase and in accelerated turnover of its substrates including IκB and β-catenin. CRD-BP is essential for the induction of both βTrCP1 and c-Myc by β-catenin signalling in colorectal cancer cells. High levels of CRD-BP that are found in primary human colorectal tumours exhibiting active β-catenin/Tcf signalling implicates CRD-BP induction in the upregulation of βTrCP1, in the activation of dimeric transcription factor NF-κB and in the suppression of apoptosis in these cancers.

Wnt Signaling Stimulates Transcriptional Outcome of the Hedgehog Pathway by Stabilizing GLI1 mRNA

Wnt and Hedgehog signaling pathways play central roles in embryogenesis, stem cell maintenance, and tumorigenesis. However, the mechanisms by which these two pathways interact are not well understood. Here, we identified a novel mechanism by which Wnt signaling pathway stimulates the transcriptional output of Hedgehog signaling. Wnt/beta-catenin signaling induces expression of an RNA-binding protein, CRD-BP, which in turn binds and stabilizes GLI1 mRNA, causing an elevation of GLI1 expression and transcriptional activity. The newly described mode of regulation of GLI1 seems to be important to several functions of Wnt, including survival and proliferation of colorectal cancer cells.

IMP1 promotes tumor growth, dissemination and a tumor-initiating cell phenotype in colorectal cancer cell xenografts

Igf2 mRNA binding protein 1 (IMP1, CRD-BP, ZBP-1) is a messenger RNA binding protein that we have shown previously to regulate colorectal cancer (CRC) cell growth in vitro. Furthermore, increased IMP1 expression correlates with enhanced metastasis and poor prognosis in CRC patients. In the current study, we sought to elucidate IMP1-mediated functions in CRC pathogenesis in vivo. Using CRC cell xenografts, we demonstrate that IMP1 overexpression promotes xenograft tumor growth and dissemination into the blood. Furthermore, intestine-specific knockdown of Imp1 dramatically reduces tumor number in the Apc (Min/+) mouse model of intestinal tumorigenesis. In addition, IMP1 knockdown xenografts exhibit a reduced number of tumor cells entering the circulation, suggesting that IMP1 may directly modulate this early metastatic event. We further demonstrate that IMP1 overexpression decreases E-cadherin expression, promotes survival of single tumor cell-derived colonospheres and promotes enrichment and maintenance of a population of CD24+CD44+ cells, signifying that IMP1 overexpressing cells display evidence of loss of epithelial identity and enhancement of a tumor-initiating cell phenotype. Taken together, these findings implicate IMP1 as a modulator of tumor growth and provide evidence for a novel role of IMP1 in early events in CRC metastasis.

Transcriptional Regulation of CRD-BP by c-myc Implications for c-myc Functions

The coding region determinant binding protein, CRD-BP, is a multifunctional RNA binding protein involved in different processes such as mRNA turnover, translation control, and localization. It is mostly expressed in fetal and neonatal tissues, where it regulates many transcripts essential for normal embryonic development. CRD-BP is scarce or absent in normal adult tissues but reactivated and/or overexpressed in various neoplastic and preneoplastic tumors and in most cell lines. Its expression has been associated with the most aggressive form of some cancers. CRD-BP is an important regulator of different genes including a variety of oncogenes or proto-oncogenes (c-myc, β-TrCP1, GLI1, etc.). Regulation of CRD-BP expression is critical for proper control of its targets as its overexpression may play an important role in abnormal cell proliferation, suppression of apoptosis, invasion, and metastasis. Molecular bases of the regulatory mechanisms governing CRD-BP expression are still not completely elucidated. In this article, we have identified c-myc as a novel transcriptional regulator of CRD-BP. We show that c-myc binds to CRD-BP promoter and induces its transcription. This induction of CRD-BP expression contributes to the role of c-myc in the regulation of translation, increase in cell size, and acceleration of cell cycle progression via a mechanism involving upregulation of β-TrCP1 levels and activities and accelerated degradation of PDCD4.

Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis

Although cancer cell fusion has been suggested as a mechanism of cancer metastasis, the underlying mechanisms defining this process are poorly understood. In a recent study, apoptotic cells were newly identified as a type of cue that induces signaling via phosphatidylserine receptors to promote fusion of myoblasts. The microenvironment of breast tumors is often hypoxic, and because apoptosis is greatly increased in hypoxic conditions, we decided to investigate whether the mechanism of breast cancer cell fusion with mesenchymal stem/multipotent stromal cells (MSCs) involves apoptosis. We used a powerful tool for identification and tracking of hybrids based on bimolecular fluorescence complementation (BiFC) and found that breast cancer cells fused spontaneously with MSCs. This fusion was significantly enhanced with hypoxia and signaling associated with apoptotic cells, especially between nonmetastatic breast cancer cells and MSCs. In addition, the hybrids showed a significantly higher migratory capacity than did the parent cells. Taken together, these findings describe a mechanism by which hypoxiainduced apoptosis stimulates fusion between MSCs and breast tumor cells resulting in hybrids with an enhanced migratory capacity that may enable their dissemination to distant sites or metastases. In the long run, this study may provide new strategies for developing novel drugs for preventing cancer metastasis.—Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. Apoptosis‐induced cancer cell fusion: a mechanism of breast cancer metastasis. FASEB J. 29, 4036‐4045 (2015). www.fasebj.org

Role of CRD-BP in the Growth of Human Basal Cell Carcinoma Cells

Although the number of new cases of Basal Cell Carcinoma (BCC) has increased rapidly in the last few decades, the molecular basis of its pathogenesis is not completely understood. Activation of Hedgehog (Hh) signaling pathway has been shown to be a key factor driving the development of BCC. The Wnt/β-catenin signaling pathway was also shown to be activated in BCCs and to perhaps modulate the activity of Hh pathway. We have previously identified a novel mechanism by which Wnt signaling regulates the transcriptional outcome of Hh signaling pathway. We demonstrated that CRD-BP, a direct target of the Wnt/β-catenin signaling, binds to GLI1 mRNA, stabilizes it, and consequently upregulates its levels (mRNA and protein) and activities. We hypothesized that Wnt-induced and CRD-BP-dependent regulation of GLI1 expression and activities is important to the development of BCC. In this study, we show that CRD-BP is over-expressed in BCC and that its expression positively correlates with the activation of both Wnt and Hh signaling pathways. We also describe the generation and characterization of a human BCC cell line. This cell line was utilized to demonstrate the importance of CRD-BP-dependent regulation of GLI1 expression and activities in the development of BCC.

Prodifferentiation, Anti-Inflammatory and Antiproliferative Effects of Delphinidin, a Dietary Anthocyanidin, in a Full-Thickness Three-Dimensional Reconstituted Human Skin Model of Psoriasis

Background: Psoriasis is a chronic inflammatory disorder of skin and joints for which conventional treatments that are effective in clearing the moderate-to-severe disease are limited due to long-term safety issues. This necessitates exploring the usefulness of botanical agents for treating psoriasis. We previously showed that delphinidin, a diet-derived anthocyanidin endowed with antioxidant and anti-inflammatory properties, induces normal epidermal keratinocyte differentiation and suggested its possible usefulness for the treatment of psoriasis [1]. Objectives: To investigate the effect of delphinidin (0-20 μM; 2-5 days) on psoriatic epidermal keratinocyte differentiation, proliferation and inflammation using a three-dimensional reconstructed human psoriatic skin equivalent (PSE) model. Methods: PSEs and normal skin equivalents (NSEs) established on fibroblast-contracted collagen gels with respective psoriatic and normal keratinocytes and treated with/without delphinidin were analyzed for histology, expression of markers of differentiation, proliferation and inflammation using histomorphometry, immunoblotting, immunochemistry, qPCR and cultured supernatants for cytokine with a Multi-Analyte ELISArray Kit. Results: Our data show that treatment of PSE with delphinidin induced (1) cornification without affecting apoptosis and (2) the mRNA and protein expression of markers of differentiation (caspase-14, filaggrin, loricrin, involucrin). It also decreased the expression of markers of proliferation (Ki67 and proliferating cell nuclear antigen) and inflammation (inducible nitric oxide synthase and antimicrobial peptides S100A7-psoriasin and S100A15-koebnerisin, which are often induced in psoriatic skin). ELISArray showed increased release of psoriasis-associated keratinocyte-derived proinflammatory cytokines in supernatants of the PSE cultures, and this increase was significantly suppressed by delphinidin. Conclusions: These observations provide a rationale for developing delphinidin for the management of psoriasis.

Cancer Cell Fusion: Mechanisms Slowly Unravel.

Although molecular mechanisms and signaling pathways driving invasion and metastasis have been studied for many years, the origin of the population of metastatic cells within the primary tumor is still not well understood. About a century ago, Aichel proposed that cancer cell fusion was a mechanism of cancer metastasis. This hypothesis gained some support over the years, and recently became the focus of many studies that revealed increasing evidence pointing to the possibility that cancer cell fusion probably gives rise to the metastatic phenotype by generating widespread genetic and epigenetic diversity, leading to the emergence of critical populations needed to evolve resistance to the treatment and development of metastasis. In this review, we will discuss the clinical relevance of cancer cell fusion, describe emerging mechanisms of cancer cell fusion, address why inhibiting cancer cell fusion could represent a critical line of attack to limit drug resistance and to prevent metastasis, and suggest one new modality for doing so.

Loss of Stromal IMP1 Promotes a Tumorigenic Microenvironment in the Colon.

The colon tumor microenvironment is becoming increasingly recognized as a complex but central player in the development of many cancers. Previously, we identified an oncogenic role for the mRNA-binding protein IMP1 (IGF2BP1) in the epithelium during colon tumorigenesis. In the current study, we reveal the contribution of stromal IMP1 in the context of colitis-associated colon tumorigenesis. Interestingly, stromal deletion of Imp1 (Dermo1Cre;Imp1LoxP/LoxP, or Imp1ΔMes) in the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer resulted in increased tumor numbers of larger size and more advanced histologic grade than controls. In addition, Imp1ΔMes mice exhibited a global increase in protumorigenic microenvironment factors, including enhanced inflammation and stromal components. Evaluation of purified mesenchyme from AOM/DSS-treated Imp1ΔMes mice demonstrated an increase in hepatocyte growth factor (HGF), which has not been associated with regulation via IMP1. Genetic knockdown of Imp1 in human primary fibroblasts confirmed an increase in HGF with Imp1 loss, demonstrating a specific, cell-autonomous role for Imp1 loss to increase HGF expression. Taken together, these data demonstrate a novel tumor-suppressive role for IMP1 in colon stromal cells and underscore an exquisite, context-specific function for mRNA-binding proteins, such as IMP1, in disease states. Implications: The tumor-suppressive role of stromal IMP1 and its ability to modulate protumorigenic factors suggest that IMP1 status is important for the initiation and growth of epithelial tumors. Mol Cancer Res; 13(11); 1478–86. ©2015 AACR. See related article by Koltsova and Grivennikov, p. 1452

An in vitro inverted vertical invasion assay to avoid manipulation of rare or sensitive cell types

The ability to quantify cell migration and invasion is critical in the study of cancer metastasis. Current invasion assays, such as the Boyden Chamber, present difficulties in the measurement of the invasion of cells that are few in number and are intrinsically tied to the cell microenvironment. There exists a need for a three-dimensional invasion assay that is easily reproduced, accessible for most laboratories, and requires no displacement of cells from their original microenvironment. Here we present a simple design for an inverted vertical invasion assay able to assess the invasion capabilities of cells in a three dimensional, extracellular matrix-based environment without displacement from the original culture location. We used the assay to determine the migratory capacity of hybrids between mesenchymal/multipotent stem/stroma cells (MSCs) and breast cancer cells MCF7. These hybrids are formed reliably but rarely (1 in 1,000 cells) and for this reason require an invasion assay that does not involve extensive cell manipulation. Using this assay, we found that MSCs, breast cancer cells, and corresponding fusion products are able to migrate and invade through the extracellular matrix and that hybrids invade in a manner more similar to stromal cells than cancer cells. Thus, this assay can aid the study of the invasive capacity of both cancerous cells and associated fusion hybrids and could augment testing of therapeutic strategies to inhibit metastatic spread.