Stabilization of E-cadherin adhesions by COX-2/GSK3ß signaling is a targetable pathway in metastatic breast cancer.

Metastatic progression of epithelial cancers can be associated with epithelial-mesenchymal transition (EMT) including transcriptional inhibition of E-cadherin (CDH1) expression. Recently, EM plasticity (EMP) and E-cadherin-mediated, cluster-based metastasis and treatment resistance have become more appreciated. However, the mechanisms that maintain E-cadherin expression in this context are less understood. Through studies of inflammatory breast cancer (IBC) and a 3D tumor cell "emboli" culture paradigm, we discovered that cyclooxygenase 2 (COX-2; PTGS2), a target gene of C/EBPδ (CEBPD), or its metabolite prostaglandin E2 (PGE2) promotes protein stability of E-cadherin, ß-catenin, and p120 catenin through inhibition of GSK3ß. The COX-2 inhibitor celecoxib downregulated E-cadherin complex proteins and caused cell death. Coexpression of E-cadherin and COX-2 was seen in breast cancer tissues from patients with poor outcome and, along with inhibitory GSK3ß phosphorylation, in patient-derived xenografts (PDX) including triple negative breast cancer (TNBC).Celecoxib alone decreased E-cadherin protein expression within xenograft tumors, though CDH1 mRNA levels increased, and reduced circulating tumor cell (CTC) clusters. In combination with paclitaxel, celecoxib attenuated or regressed lung metastases. This study has uncovered a mechanism by which metastatic breast cancer cells can maintain E-cadherin-mediated cell-to-cell adhesions and cell survival, suggesting that some patients with COX-2+/E-cadherin+ breast cancer may benefit from targeting of the PGE2 signaling pathway.

PERK signaling through C/EBPδ contributes to ER stress-induced expression of immunomodulatory and tumor promoting chemokines by cancer cells.

Cancer cells experience endoplasmic reticulum (ER) stress due to activated oncogenes and conditions of nutrient deprivation and hypoxia. The ensuing unfolded protein response (UPR) is executed by ATF6, IRE1 and PERK pathways. Adaptation to mild ER stress promotes tumor cell survival and aggressiveness. Unmitigated ER stress, however, will result in cell death and is a potential avenue for cancer therapies. Because of this yin-yang nature of ER stress, it is imperative that we fully understand the mechanisms and dynamics of the UPR and its contribution to the complexity of tumor biology. The PERK pathway inhibits global protein synthesis while allowing translation of specific mRNAs, such as the ATF4 transcription factor. Using thapsigargin and tunicamycin to induce acute ER stress, we identified the transcription factor C/EBPδ (CEBPD) as a mediator of PERK signaling to secretion of tumor promoting chemokines. In melanoma and breast cancer cell lines, PERK mediated early induction of C/EBPδ through ATF4-independent pathways that involved at least in part Janus kinases and the STAT3 transcription factor. Transcriptional profiling revealed that C/EBPδ contributed to 20% of thapsigargin response genes including chaperones, components of ER-associated degradation, and apoptosis inhibitors. In addition, C/EBPδ supported the expression of the chemokines CXCL8 (IL-8) and CCL20, which are known for their tumor promoting and immunosuppressive properties. With a paradigm of short-term exposure to thapsigargin, which was sufficient to trigger prolonged activation of the UPR in cancer cells, we found that conditioned media from such cells induced cytokine expression in myeloid cells. In addition, activation of the CXCL8 receptor CXCR1 during thapsigargin exposure supported subsequent sphere formation by cancer cells. Taken together, these investigations elucidated a novel mechanism of ER stress-induced transmissible signals in tumor cells that may be particularly relevant in the context of pharmacological interventions.

The STAT3 inhibitor Stattic acts independently of STAT3 to decrease histone acetylation and modulate gene expression.

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.

Slug and E-Cadherin: Stealth Accomplices?

During physiological epithelial-mesenchymal transition (EMT), which is important for embryogenesis and wound healing, epithelial cells activate a program to remodel their structure and achieve a mesenchymal fate. In cancer cells, EMT confers increased invasiveness and tumor-initiating capacity, which contribute to metastasis and resistance to therapeutics. However, cellular plasticity that navigates between epithelial and mesenchymal states and maintenance of a hybrid or partial E/M phenotype appears to be even more important for cancer progression. Besides other core EMT transcription factors, the well-characterized Snail-family proteins Snail (SNAI1) and Slug (SNAI2) play important roles in both physiological and pathological EMT. Often mentioned in unison, they do, however, differ in their functions in many scenarios. Indeed, Slug expression does not always correlate with complete EMT or loss of E-cadherin (CDH1). For example, Slug plays important roles in mammary epithelial cell progenitor cell lineage commitment and differentiation, DNA damage responses, hematopoietic stem cell self-renewal, and in pathologies such as pulmonary fibrosis and atherosclerosis. In this Perspective, we highlight Slug functions in mammary epithelial cells and breast cancer as a "non-EMT factor" in basal epithelial cells and stem cells with focus reports that demonstrate co-expression of Slug and E-cadherin. We speculate that Slug and E-cadherin may cooperate in normal mammary gland and breast cancer/stem cells and advocate for functional assessment of such Slug+/E-cadherinlow/+ (SNAI2+/CDH1low/+) "basal-like epithelial" cells. Thus, Slug may be regarded as less of an EMT factor than driver of the basal epithelial cell phenotype.

Cyclometalated rhodium and iridium complexes with imidazole containing Schiff bases: Synthesis, structure and cellular imaging.

Cyclometalated rhodium(III) and iridium(III) complexes (1-4) of two Schiff base ligands L1 and L2 with the general formula [M(ppy)2(Ln)]Cl {M = Rh, Ir; ppy = 2-phenylpyridine; n = 1, 2; L = Schiff base ligand} have been synthesized. The new ligands and the complexes have been characterized with spectroscopic techniques. Electrochemistry of the complexes revealed anodic behavior, corresponding to an M(III) to M(IV) oxidation. The X-ray crystal structures of complexes 2 and 4 have also been determined to interpret the coordination behavior of the complexes. Photophysical study shows that all the complexes display fluorescence at room temperature with quantum yield of about 3 × 10-2 to 5 × 10-2. The electronic absorption spectra of all the complexes fit well with the computational studies. Cellular imaging studies were done with the newly synthesized complexes. To the best of our knowledge, this is the first report of organometallic complexes of rhodium(III) and iridium(III) with Schiff base ligands explored for cellular imaging. Emphasis of this work lies on the structural features, photophysical behavior, cellular uptake and imaging of the fluorescent transition metal complexes.

Development of a cyclometalated iridium complex with specific intramolecular hydrogen-bonding that acts as a fluorescent marker for the endoplasmic reticulum and causes photoinduced cell death.

Cyclometalated iridium complexes have important applications as phosphorescent probes for cellular imaging due to their photophysical properties. Moreover, these properties also make them potential candidates as photosensitizers for photodynamic therapy (PDT) of tumors and skin diseases. Treatment of MCF7 breast carcinoma cells with a heteroleptic phosphorescent cyclometalated iridium(III) complex C2 followed by confocal imaging indicates that the complex selectively localizes and exhibits high fluorescence in the endoplasmic reticulum. In an unprecedented approach, systematic alteration of functional groups or the metal core in C2 to synthesize a series of iridium(III) complexes (C1­C10) and an organometallic rhenium complex C11 with an imidazolyl modified phenanthroline ligand has indicated the functional groups and their interactions that are responsible for this selective localization. Remarkably, the exposure of the cells treated with C2 to irradiation at 405 nm for one hour led to membrane blebbing and cell death, demonstrating a photosensitizing property of the compound.