Overexpression of REST Represses the Epithelial-Mesenchymal Transition Process and Decreases the Aggressiveness of Prostate Cancer Cells.

The RE-1 silencing transcription factor (REST) is a repressor factor related to neuroendocrine prostate cancer (PCa) (NEPC), a poor prognostic stage mainly associated with castration-resistant PCa (CRPC). NEPC is associated with cell transdifferentiation and the epithelial-mesenchymal transition (EMT) in cells undergoing androgen deprivation therapy (ADT) and enzalutamide (ENZ). The effect of REST overexpression in the 22rv1 cell line (xenograft-derived prostate cancer) on EMT, migration, invasion, and the viability for ENZ was evaluated. EMT genes, Twist and Zeb1, and the androgen receptor (AR) were evaluated through an RT-qPCR and Western blot in nuclear and cytosolic fractions of REST-overexpressing 22rv1 cells (22rv1-REST). The migratory and invasive capacities of 22rv1-REST cells were evaluated via Transwell® assays with and without Matrigel, respectively, and their viability for enzalutamide via MTT assays. The 22rv1-REST cells showed decreased nuclear levels of Twist, Zeb1, and AR, and a decreased migration and invasion and a lower viability for ENZ compared to the control. Results were expressed as the mean + SD of three independent experiments (Mann-Whitney U test, Kruskal-Wallis, Tukey test). REST behaves like a tumor suppressor, decreasing the aggressiveness of 22rv1 cells, probably through the repression of EMT and the neuroendocrine phenotype. Furthermore, REST could represent a response marker to ENZ in PCa patients.

The Cytoplasmic Region of SARAF Reduces Triple-Negative Breast Cancer Metastasis through the Regulation of Store-Operated Calcium Entry.

Triple-negative breast cancer has a poor prognosis and is non-responsive to first-line therapies; hence, new therapeutic strategies are needed. Enhanced store-operated Ca2+ entry (SOCE) has been widely described as a contributing factor to tumorigenic behavior in several tumor types, particularly in breast cancer cells. SOCE-associated regulatory factor (SARAF) acts as an inhibitor of the SOCE response and, therefore, can be a potential antitumor factor. Herein, we generated a C-terminal SARAF fragment to evaluate the effect of overexpression of this peptide on the malignancy of triple-negative breast cancer cell lines. Using both in vitro and in vivo approaches, we showed that overexpression of the C-terminal SARAF fragment reduced proliferation, cell migration, and the invasion of murine and human breast cancer cells by decreasing the SOCE response. Our data suggest that regulating the activity of the SOCE response via SARAF activity might constitute the basis for further alternative therapeutic strategies for triple-negative breast cancer.

Regulation of the Intestinal Extra-Adrenal Steroidogenic Pathway Component LRH-1 by Glucocorticoids in Ulcerative Colitis.

Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) and can be treated with glucocorticoids (GC), although some patients are unresponsive to this therapy. The transcription factor LRH-1/NR5A2 is critical to intestinal cortisol production (intestinal steroidogenesis), being reduced in UC patients. However, the relationship between LRH-1 expression and distribution with altered corticosteroid responses is unknown. To address this, we categorized UC patients by their steroid response. Here, we found that steroid-dependent and refractory patients presented reduced glucocorticoid receptor (GR)-mediated intestinal steroidogenesis compared to healthy individuals and responder patients, possibly related to increased colonic mucosa GR isoform beta (GRß) content and cytoplasmic LRH-1 levels in epithelial and lamina propria cells. Interestingly, an intestinal epithelium-specific GR-induced knockout (GRiKO) dextran sodium sulfate (DSS)-colitis mice model presented decreased epithelial LRH-1 expression, whilst it increased in the lamina propria compared to DSS-treated control mice. Mechanistically, GR directly induced NR5A2 gene expression in CCD841CoN cells and human colonic organoids. Furthermore, GR bound to two glucocorticoid-response elements within the NR5A2 promoter in dexamethasone-stimulated CCD841CoN cells. We conclude that GR contributes to intestinal steroidogenesis by inducing LRH-1 in epithelial cells, suggesting LRH-1 as a potential marker for glucocorticoid-impaired response in UC. However, further studies with a larger patient cohort will be necessary to confirm role of LRH-1 as a therapeutic biomarker.

TRP Channels Interactome as a Novel Therapeutic Target in Breast Cancer.

Breast cancer is one of the most frequent cancer types worldwide and the first cause of cancer-related deaths in women. Although significant therapeutic advances have been achieved with drugs such as tamoxifen and trastuzumab, breast cancer still caused 627,000 deaths in 2018. Since cancer is a multifactorial disease, it has become necessary to develop new molecular therapies that can target several relevant cellular processes at once. Ion channels are versatile regulators of several physiological- and pathophysiological-related mechanisms, including cancer-relevant processes such as tumor progression, apoptosis inhibition, proliferation, migration, invasion, and chemoresistance. Ion channels are the main regulators of cellular functions, conducting ions selectively through a pore-forming structure located in the plasma membrane, protein-protein interactions one of their main regulatory mechanisms. Among the different ion channel families, the Transient Receptor Potential (TRP) family stands out in the context of breast cancer since several members have been proposed as prognostic markers in this pathology. However, only a few approaches exist to block their specific activity during tumoral progress. In this article, we describe several TRP channels that have been involved in breast cancer progress with a particular focus on their binding partners that have also been described as drivers of breast cancer progression. Here, we propose disrupting these interactions as attractive and potential new therapeutic targets for treating this neoplastic disease.

TRP Channels Regulation of Rho GTPases in Brain Context and Diseases.

Neurological and neuropsychiatric disorders are mediated by several pathophysiological mechanisms, including developmental and degenerative abnormalities caused primarily by disturbances in cell migration, structural plasticity of the synapse, and blood-vessel barrier function. In this context, critical pathways involved in the pathogenesis of these diseases are related to structural, scaffolding, and enzymatic activity-bearing proteins, which participate in Ca2+- and Ras Homologs (Rho) GTPases-mediated signaling. Rho GTPases are GDP/GTP binding proteins that regulate the cytoskeletal structure, cellular protrusion, and migration. These proteins cycle between GTP-bound (active) and GDP-bound (inactive) states due to their intrinsic GTPase activity and their dynamic regulation by GEFs, GAPs, and GDIs. One of the most important upstream inputs that modulate Rho GTPases activity is Ca2+ signaling, positioning ion channels as pivotal molecular entities for Rho GTPases regulation. Multiple non-selective cationic channels belonging to the Transient Receptor Potential (TRP) family participate in cytoskeletal-dependent processes through Ca2+-mediated modulation of Rho GTPases. Moreover, these ion channels have a role in several neuropathological events such as neuronal cell death, brain tumor progression and strokes. Although Rho GTPases-dependent pathways have been extensively studied, how they converge with TRP channels in the development or progression of neuropathologies is poorly understood. Herein, we review recent evidence and insights that link TRP channels activity to downstream Rho GTPase signaling or modulation. Moreover, using the TRIP database, we establish associations between possible mediators of Rho GTPase signaling with TRP ion channels. As such, we propose mechanisms that might explain the TRP-dependent modulation of Rho GTPases as possible pathways participating in the emergence or maintenance of neuropathological conditions.

SNAIL expression correlates with the translocation of syndecan­1 intracellular domain into the nucleus in prostate cancer cell lines.

Zinc finger protein SNAI1 (SNAIL) and zinc finger protein SNAI2 (SLUG) transcription factors promote epithelial­mesenchymal transition, a process through which epithelial cells acquire a mesenchymal phenotype, increasing their migratory and invasive properties. In prostate cancer (PCa) progression, increased expression levels of SNAIL and SLUG have been described. In advanced PCa, a decrease in the cell surface proteoglycan syndecan­1 (SDC­1), which has a role in cell­to­extracellular matrix adhesion, has been observed. Notably, SDC­1 nuclear location has been observed in mesenchymal cancers. The present study aimed to determine if SNAIL and SLUG may be associated with the nuclear location of SDC­1 in PCa. To determine the location of SDC­1, antibodies against its intracellular domain (ID) or extracellular domain (ED) were used in benign prostatic hyperplasia (BPH) and PCa samples with high Gleason scores. Only ID­SDC­1 was located in the cell nuclei in advanced PCa samples, but not in the BPH samples. ED­SDC­1 was located in the cell membrane and cytoplasm, exhibiting decreased levels in PCa in comparison with those in BPH. Furthermore, LNCaP and PC3 PCa cell lines with ectopic SNAIL expression exhibited nuclear ID­SDC­1. No change was observed in the ED­SDC­1 levels, and maintained its location in the cell membrane and cytoplasm. SLUG induced no change in ID­SDC­1 location. At the protein level, an association between SNAIL and nuclear ID­SDC­1 was observed. In conclusion, the results of the present study demonstrated that nuclear ID­SDC­1 localization was associated with SNAIL expression in PCa cell lines.

Corrigendum: Interleukin 33/ST2 Axis Components Are Associated to Desmoplasia, a Metastasis-Related Factor in Colorectal Cancer.

[This corrects the article DOI: 10.3389/fimmu.2019.01394.].

Interleukin 33/ST2 Axis Components Are Associated to Desmoplasia, a Metastasis-Related Factor in Colorectal Cancer.

In colorectal cancer (CRC), cancer-associated fibroblasts (CAFs) are the most abundant component from the tumor microenvironment (TM). CAFs facilitate tumor progression by inducing angiogenesis, immune suppression and invasion, thus altering the organization/composition of the extracellular matrix (i.e., desmoplasia) and/or activating epithelial-mesenchymal transition (EMT). Soluble factors from the TM can also contribute to cell invasion through secretion of cytokines and recently, IL-33/ST2 pathway has gained huge interest as a protumor alarmin, promoting progression to metastasis by inducing changes in TM. Hence, we analyzed IL-33 and ST2 content in tumor and healthy tissue lysates and plasma from CRC patients. Tissue localization and distribution of these molecules was evaluated by immunohistochemistry (using localization reference markers α-smooth muscle actin or α-SMA and E-cadherin), and clinical/histopathological information was obtained from CRC patients. In vitro experiments were conducted in primary cultures of CAFs and normal fibroblasts (NFs) isolated from tumor and healthy tissue taken from CRC patients. Additionally, migration and proliferation analysis were performed in HT29 and HCT116 cell lines. It was found that IL-33 content increases in left-sided CRC patients with lymphatic metastasis, with localization in tumor epithelia associated with abundant desmoplasia. Although ST2 content showed similarities between tumor and healthy tissue, a decreased immunoreactivity was observed in left-sided tumor stroma, associated to metastasis related factors (advanced stages, abundant desmoplasia, and presence of tumor budding). A principal component analysis (including stromal and epithelial IL-33/ST2 and α-SMA immunoreactivity with extent of desmoplasia) allowed us to distinguish clusters of low, intermediate and abundant desmoplasia, with potential to develop a diagnostic signature with benefits for further therapeutic targets. IL-33 transcript levels from CAFs directly correlated with CRC cell line migration induced by CAFs conditioned media, with rhIL-33 inducing a mesenchymal phenotype in HT29 cells. These results indicate a role of IL-33/ST2 in tumor microenvironment, specifically in the interaction between CAFs and epithelial tumor cells, thus contributing to invasion and metastasis in left-sided CRC, most likely by activating desmoplasia.

The transcription factor ZEB1 promotes chemoresistance in prostate cancer cell lines.

One of the factors promoting tumoral progress is the abnormal activation of the epithelial-mesenchymal transition (EMT) program which has been associated with chemoresistance in tumoral cells. The transcription factor zinc finger E-box-binding homeobox 1 (ZEB1), a key EMT activator, has recently been related to docetaxel resistance, the main chemotherapeutic used in advanced prostate cancer treatment. The mechanisms involved in this protective effect are still unclear. In a previous work, we demonstrated that ZEB1 expression induced an EMT-like phenotype in prostate cancer cell lines. In this work, we used prostate cancer cell lines 22Rv1 and DU145 to study the effect of ZEB1 modulation on docetaxel resistance and its possible mechanisms. The results showed that ZEB1 overexpression conferred to 22Rv1 cell resistance to docetaxel while its silencing made DU145 cells more sensitive to it. Analysis of resistance markers showed no presence of ATP-binding cassette subfamily B member 1 (MDR1) and no changes in breast cancer resistance protein (BCRP) or ATP-binding cassette subfamily C member 10 (MRP7). However, a correlation between ZEB1, multidrug resistance-associated protein 1 (MRP1), and ATP-binding cassette subfamily C member 4 (MRP4) expression was observed. MRP4 inhibition, using MK571, resensitized cells with ZEB1 overexpression to docetaxel treatment. In addition, modulation of ZEB1 and subsequent change in MRP4 expression correlated with a lower apoptotic response to docetaxel, characterized by lower B-cell lymphoma 2 (Bcl2), high BCL2-associated X protein (Bax), and high active caspase 3 expression. The response to docetaxel in our model seems to be mediated mainly by activation of the apoptotic death program. Our results showed that modulation of MRP4 could be a mediator of ZEB1-related resistance to docetaxel in prostate cancer, making it a possible marker for chemotherapy response in patients who do not express MDR1.

The transcription factor ZEB1 promotes an aggressive phenotype in prostate cancer cell lines.

It has been reported that one of the factors that promotes tumoral progression is the abnormal activation of the epithelial-mesenchymal transition program. This process is associated with tumoral cells acquiring invasive and malignant properties and has the transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) as one of its main activators. However, the role of ZEB1 in promoting malignancy in prostate cancer (PCa) is still unclear. Here, we report that ZEB1 expression correlates with Gleason score in PCa samples and that expression of ZEB1 regulates epithelial-mesenchymal transition and malignant characteristics in PCa cell lines. The results showed that ZEB1 expression is higher in samples of higher malignancy and that overexpression of ZEB1 was able to induce epithelial-mesenchymal transition by upregulating the mesenchymal marker Vimentin and downregulating the epithelial marker E-Cadherin. On the contrary, ZEB1 silencing repressed Vimentin expression and upregulated E-Cadherin. ZEB1 expression conferred enhanced motility and invasiveness and a higher colony formation capacity to 22Rv1 cells whereas DU145 cells with ZEB1 silencing showed a decrease in those same properties. The results showed that ZEB1 could be a key promoter of tumoral progression toward advanced stages of PCa.