Nutrition and Breast Cancer Research in Arab Countries: Gaps, Opportunities, and Recommendations.

According to the WHO, Arab countries have the highest relative increase in Breast Cancer (BC) rates worldwide. Current shifts in dietary patterns in these countries are postulated as important modifiable risk factors of the disease. The objectives of this review were to examine the gaps and opportunities in the extent, range and nature of nutrition-related BC research in Arab countries. Studies (n = 286) were identified through searching 14 electronic databases. Among the gaps identified were limited international collaborations, preponderance of laboratory-based research at the expense of population-based research, focus on single supplement/nutrient/food research, limited use of dietary assessment tools, and studying nutrition in isolation of other environmental factors. Despite these gaps, several opportunities appeared. The distribution of papers among Arab countries suggested that collaboration between high and middle income countries could create a positive synergy between research expertise and wealth. In addition, the steady increase in the number of articles published during the last two decades reflected a promising momentum in nutrition and BC research in the Arab world. These gaps and opportunities constituted context-specific evidence to orient nutrition and BC research in Arab countries which could ultimately lead to development of effective interventions for prevention of BC in these countries.

A Signature of Four Circulating microRNAs as Potential Biomarkers for Diagnosing Early-Stage Breast Cancer.

Breast cancer (BC) is the most predominant type of cancer among women. The aim of this study is to find new biomarkers that can help in early detection of BC, especially for those who are too young to be screened using mammography as per guidelines. Using microRNA microarray, we previously showed dysregulation of 74 microRNAs in tumors from early BC patients as compared with normal adjacent tissues, which we were interested in studying in blood circulation. In this study, we investigated the expression of 12 microRNA (miR-21/miR-155/miR-23a/miR-130a/miR-145/miR-425-5p/miR-139-5p/miR-451/miR-195/miR-125b/miR-100, and miR-182) in the plasma of 41 newly diagnosed Lebanese BC patients with early invasive ductal carcinoma as compared with 32 healthy controls. Total RNA was extracted from plasma, and expression levels of miRNA of interest were measured using RT-qPCR followed by statistical analysis; miR-21, miR-155, miR-23a, miR-130a, miR-145, miR-425-5p, and miR-139-5p were significantly upregulated and miR-451 was significantly downregulated, in the plasma of BC patients as compared with healthy controls. The positively correlated miR-23a, miR-21, and miR-130a had a high diagnostic accuracy (86%). Importantly, the combination of miR-145/miR-425-5p/miR-139-5p/miR-130a scored the highest diagnostic accuracy of 95% with AUC = 0.97 (sensitivity 97% and specificity 91%). MicroRNAs are promising non-invasive diagnostic biomarkers for early-stage BC with the panel of miR-145/miR-425-5p/miR-139-5p/miR-130a having the highest diagnostic accuracy.

Gap Junctions and Wnt Signaling in the Mammary Gland: a Cross-Talk?

Connexins (Cxs), the building blocks of gap junctions (GJs), exhibit spatiotemporal patterns of expression and regulate the development and differentiation of the mammary gland, acting via channel-dependent and channel-independent mechanisms. Impaired Cx expression and localization are reported in breast cancer, suggesting a tumor suppressive role for Cxs. The signaling events that mediate the role of GJs in the development and tumorigenesis of the mammary gland remain poorly identified. The Wnt pathways, encompassing the canonical or the Wnt/ß-catenin pathway and the noncanonical ß-catenin-independent pathway, also play important roles in those processes. Indeed, aberrant Wnt signaling is associated with breast cancer. Despite the coincident roles of Cxs and Wnt pathways, the cross-talk in the breast tissue is poorly defined, although this is reported in a number of other tissues. Our previous studies revealed a channel-independent role for Cx43 in inducing differentiation or suppressing tumorigenesis of mammary epithelial cells by acting as a negative regulator of the Wnt/ß-catenin pathway. Here, we provide a brief overview of mammary gland development, with emphasis on the role of Cxs in development and tumorigenesis of this tissue. We also discuss the role of Wnt signaling in similar contexts, and review the literature illustrating interplay between Cxs and Wnt pathways.

Cross-Roads to Drug Resistance and Metastasis in Breast Cancer: miRNAs Regulatory Function and Biomarker Capability.

Breast cancer and specifically metastatic breast cancer (mBC) constitutes a major health burden worldwide with the highest number of cancer-related mortality among women across the globe. Despite having similar subtypes, breast cancer patients present with a spectrum of aggressiveness and responsiveness to therapy due to cancer heterogeneity. Drug resistance and metastasis contribute to therapy failure and cancer recurrence. Research in the past two decades has focused on microRNAs (miRNAs), small endogenous non-coding RNAs, as active players in tumorigenesis, therapy resistance and metastasis and as novel non-invasive cancer biomarkers. This is due to their unique dysregulated signatures throughout tumor progression and their tumor suppressive/oncogenic roles. Identifying miRNAs signatures capable of predicting therapy response and metastatic onset in breast cancer patients might improve prognosis and offer prolonged median and relapse-free survival rate. Despite the growing reports on miRNAs as novel non-invasive biomarkers in breast cancer and as regulators of breast cancer drug resistance or metastasis, the quest on whether some miRNAs are capable of regulating both simultaneously is inevitable, yet understudied. This chapter will review the role of miRNAs as biomarkers and as active players in inducing/reversing anti-cancer drug resistance, driving/blocking metastasis or regulating both simultaneously in breast cancer.

Antitumor activities of the synthetic retinoid ST1926 in two-dimensional and three-dimensional human breast cancer models.

Despite recent advances in chemotherapy, aggressive and metastatic breast cancers remain refractory to targeted therapy and the development of novel drugs is urgently needed. Retinoids are crucial regulators of cellular proliferation, differentiation, and cell death, and have shown potent chemotherapeutic and chemopreventive properties. The major drawback of the use of all-trans retinoic acid (ATRA) in cancer therapy is disease relapse. Therefore, synthetic retinoids, specifically ST1926, have emerged as potent anticancer agents. Given the importance of the microenvironment in modulating the response of cancer cells to chemotherapeutic drugs, we investigated the antitumor activities of ST1926 in two-dimensional (2D) and different three-dimensional (3D) human breast cancer models and compared them with ATRA. We have shown that in 2D cell culture models, ATRA-resistant MCF-7 and MDA-MB-231 cells were sensitive to ST1926 at submicromolar concentrations that spared the 'normal-like' breast epithelial cells. ST1926 induced apoptosis and S-phase arrest, caused DNA damage, and downregulated the Wnt/ß-catenin pathway in breast cancer cells in 2D and 3D cell culture models. ST1926-mediated growth inhibition was independent of the retinoid receptor-signaling pathway. Long-term treatments with low submicromolar ST1926 concentrations reduced the anchorage-independent growth and decreased the sphere-forming ability of breast cancer progenitor cells in the sphere formation assay. Furthermore, ST1926 potently induced cell death of breast cancer cells under 3D conditions and spared the lumen-forming ability of normal-like breast epithelial cells. In tested 3D models, ATRA had minimal effects on the growth of breast cancer cells compared with ST1926. In summary, our results highlight the therapeutic potential of ST1926 in breast cancer and warrant its further clinical development.

Context dependent reversion of tumor phenotype by connexin-43 expression in MDA-MB231 cells and MCF-7 cells: role of ß-catenin/connexin43 association.

Connexins (Cx), gap junction (GJ) proteins, are regarded as tumor suppressors, and Cx43 expression is often down regulated in breast tumors. We assessed the effect of Cx43 over-expression in 2D and 3D cultures of two breast adenocarcinoma cell lines: MCF-7 and MDA-MB-231. While Cx43 over-expression decreased proliferation of 2D and 3D cultures of MCF-7 by 56% and 80% respectively, MDA-MB-231 growth was not altered in 2D cultures, but exhibited 35% reduction in 3D cultures. C-terminus truncated Cx43 did not alter proliferation. Untransfected MCF-7 cells formed spherical aggregates in 3D cultures, and MDA-MB-231 cells formed stellar aggregates. However, MCF-7 cells over-expressing Cx43 formed smaller sized clusters and Cx43 expressing MDA-MB-231 cells lost their stellar morphology. Extravasation ability of both MCF-7 and MDA-MB-231 cells was reduced by 60% and 30% respectively. On the other hand, silencing Cx43 in MCF10A cells, nonneoplastic human mammary cell line, increased proliferation in both 2D and 3D cultures, and disrupted acinar morphology. Although Cx43 over-expression did not affect total levels of ß-catenin, α-catenin and ZO-2, it decreased nuclear levels of ß-catenin in 2D and 3D cultures of MCF-7 cells, and in 3D cultures of MDA-MB-231 cells. Cx43 associated at the membrane with α-catenin, ß-catenin and ZO-2 in 2D and 3D cultures of MCF-7 cells, and only in 3D conditions in MDA-MB-231 cells. This study suggests that Cx43 exerts tumor suppressive effects in a context-dependent manner where GJ assembly with α-catenin, ß-catenin and ZO-2 may be implicated in reducing growth rate, invasiveness, and, malignant phenotype of 2D and 3D cultures of MCF-7 cells, and 3D cultures of MDA-MB-231 cells, by sequestering ß-catenin away from nucleus.

Organ-on-a-chip platforms as novel advancements for studying heterogeneity, metastasis, and drug efficacy in breast cancer.

Breast cancer has the highest cancer incidence rate in women worldwide. Therapies for breast cancer have shown high success rates, yet many cases of recurrence and drug resistance are still reported. Developing innovative strategies for studying breast cancer may improve therapeutic outcomes of the disease by providing better insight into the associated molecular mechanisms. A novel advancement in breast cancer research is the utilization of organ-on-a-chip (OOAC) technology to establish in vitro physiologically relevant breast cancer biomimetic models. This emerging technology combines microfluidics and tissue culturing methods to establish organ-specific micro fabricated culture models. Here, we shed light on the advantages of OOAC platforms over conventional in vivo and in vitro models in terms of mimicking tissue heterogeneity, disease progression, and facilitating pharmacological drug testing with a focus on models of the mammary gland in both normal and breast cancer states. By highlighting the various designs and applications of the breast-on-a-chip platforms, we show that the latter propose means to facilitate breast cancer-related studies and provide an efficient approach for therapeutic drug screening in vitro.

Over-expression of miR-183-5p or miR-492 triggers invasion and proliferation and loss of polarity in non-neoplastic breast epithelium.

microRNAs (miRNAs) serve as novel noninvasive cancer biomarkers. In an HMT-3522 S1 (S1) breast epithelial risk-progression three-dimensional (3D) culture model, non-neoplastic S1 cells form a fully polarized epithelium. When silenced for the gap junction and tumor suppressor Cx43, Cx43-KO-S1 cells recapitulate pre-neoplastic phenotypes observed in tissues at risk for breast cancer in vivo. To delineate the role of miRNAs in breast tumorigenesis and identify key miRNA players in breast epithelial polarity, the miRNA profile specific to Cx43 loss in Cx43-KO-S1 compared to S1 cells was sequenced, revealing 65 differentially expressed miRNAs. A comparative analysis was conducted between these miRNAs and tumor-associated miRNAs from a young Lebanese patient validation cohort. miR-183-5p, downstream of Cx43 loss, was commonly upregulated in the patient cohort and the 3D culture model. miR-492, not attributed to Cx43 loss, was only specifically up-regulated in the young Lebanese patients. Ectopic expression of either miR-183-5p or miR-492 in S1 cells, through pLenti-III-miR-GPF vectors, resulted in the formation of larger multi-layered acini devoid of lumen, with disrupted epithelial polarity, as shown by an altered localization of Cx43, ß-catenin and Scrib, and decreased nuclear circularity in 3D cultures. Enhanced proliferation and invasion capacity were also observed. Over-expression of miR-183-5p or miR-492, therefore, induces pre-neoplastic phenotypes similar to those reported upon Cx43 loss, and may act as oncomiRs and possible biomarkers of increased breast cancer risk.

Endotoxin Triggers Tumor Initiation Events in Nontumorigenic Breast Epithelial Cells and Enhances Invasion-Related Phenotype in Pretumorigenic and Tumorigenic Breast Epithelial Cells.

Inflammation is associated with the development of several cancers, including breast cancer. However, the molecular mechanisms driving breast cancer initiation or enhancement by inflammation are yet to be deciphered. Hence, we opted to investigate the role of inflammation in initiating and enhancing tumor-like phenotypes in nontumorigenic, pretumorigenic, and tumorigenic breast epithelial cells. Noncytotoxic endotoxin (ET) concentrations capable of inducing an inflammatory phenotype were determined for the different cell lines. Results showed that short-term ET exposure upregulated matrix metalloproteinase-9 (MMP-9) activity in nontumorigenic mammary epithelial cells of mouse (SCp2) and human origins (HMT-3522 S1; S1) and upregulated inflammatory mediators including nitric oxide (NO) and interleukin 1-ß in tumorigenic human breast cells (MDA-MB-231), all in a dose-dependent manner. Long-term ET treatment, but not short-term, triggered the migration of SCp2 cells, and proliferation and migration of tumorigenic human breast cells MCF-7 and MDA-MB-231. Both short- and long-term ET exposures preferentially enhanced the invasion of pretumorigenic S1-connexin 43 knockout (Cx43-KO S1) cells compared to their nontumorigenic S1 counterparts. Moreover, both ET exposures disrupted lumen formation and apicolateral distribution of ß-catenin in 3D cultures of S1 cells. In conclusion, ET treatment at concentrations that elicited inflammatory phenotype triggered tumor initiation events in nontumorigenic and pretumorigenic breast cells, and increased tumorigenicity of breast cancer cells. Our findings highlight the role of inflammation in enhancing migration, invasion, and loss of normal 3D morphology and suggest that such inflammatory insults can "add injury" to pretumorigenic and tumorigenic breast epithelial cells.

Biomimetic sulfated glycosaminoglycans maintain differentiation markers of breast epithelial cells and preferentially inhibit proliferation of cancer cells.

Glycosaminoglycans (GAG) are key elements involved in various physiological and pathological processes including cancer. Several GAG-based drugs have been developed showing significant results and potential use as cancer therapeutics. We previously reported that alginate sulfate (AlgSulf), a GAG-mimetic, reduces the proliferation of lung adenocarcinoma cells. In this study, we evaluated the preferential effect of AlgSulf on tumorigenic and nontumorigenic mammary epithelial cells in 2D, 3D, and coculture conditions. AlgSulf were synthesized with different degrees of sulfation (DSs) varying from 0 to 2.7 and used at 100 µg/mL on HMT-3522 S1 (S1) nontumorigenic mammary epithelial cells and their tumorigenic counterparts HMT-3522 T4-2 (T4-2) cells. The anti-tumor properties of AlgSulf were assessed using trypan blue and bromodeoxyuridine proliferation (BrdU) assays, immunofluorescence staining and transwell invasion assay.  Binding of insulin and epidermal growth factor (EGF) to sulfated substrates was measured using QCM-D and ELISA. In 2D, the cell growth rate of cells treated with AlgSulf was consistently lower compared to untreated controls (p<0.001) and surpassed the effect of the native GAG heparin (positive control). In 3D, AlgSulf preferentially hindered the growth rate and the invasion potential of tumorigenic T4-2 nodules while maintaining the formation of differentiated polarized nontumorigenic S1 acini. The preferential growth inhibition of tumorigenic cells by AlgSulf was confirmed in a coculture system (p<0.001). In the ELISA assay, a trend of EGF binding was detected for sulfated polysaccharides while QCM-D analysis showed negligible binding of insulin and EGF to sulfated substrates. The preferential effect mediated by the mimetic sulfated GAGs on cancer cells may in part be growth factor dependent. Our findings suggest a potential anticancer therapeutic role of AlgSulf for the development of anticancer drugs.

A risk progression breast epithelial 3D culture model reveals Cx43/hsa_circ_0077755/miR-182 as a biomarker axis for heightened risk of breast cancer initiation.

mRNA-circRNA-miRNAs axes have been characterized in breast cancer, but not as risk-assessment axes for tumor initiation in early-onset breast cancer that is increasing drastically worldwide. To address this gap, we performed circular RNA (circRNA) microarrays and microRNA (miRNA) sequencing on acini of HMT-3522 S1 (S1) breast epithelial risk-progression culture model in 3D and chose an early-stage population miRNome for a validation cohort. Nontumorigenic S1 cells form fully polarized epithelium while pretumorigenic counterparts silenced for gap junction Cx43 (Cx43-KO-S1) lose epithelial polarity, multilayer and mimic premalignant in vivo mammary epithelial morphology. Here, 121 circRNAs and 65 miRNAs were significantly dysregulated in response to Cx43 silencing in cultured epithelia and 15 miRNAs from the patient cohort were involved in epithelial polarity disruption. Focusing on the possible sponging activity of the validated circRNAs to their target miRNAs, we found all miRNAs to be highly enriched in cancer-related pathways and cross-compared their dysregulation to actual miRNA datasets from the cultured epithelia and the patient validation cohort. We present the involvement of gap junction in post-transcriptional axes and reveal Cx43/hsa_circ_0077755/miR-182 as a potential biomarker signature axis for heightened-risk of breast cancer initiation, and that its dysregulation patterns might predict prognosis along breast cancer initiation and progression.

Gap junctional intercellular communication in hypoxia-ischemia-induced neuronal injury.

Brain hypoxia-ischemia is a relatively common and serious problem in neonates and in adults. Its consequences include long-term histological and behavioral changes and reduction in seizure threshold. Gap junction intercellular communication is pivotal in the spread of hypoxia-ischemia related injury and in mediating its long-term effects. This review provides a comprehensive and critical review of hypoxia-ischemia and hypoxia in the brain and the potential role of gap junctions in the spread of the neuronal injury induced by these insults. It also presents the effects of hypoxia-ischemia and of hypoxia on the state of gap junctions in vitro and in vivo. Understanding the mechanisms involved in gap junction-mediated neuronal injury due to hypoxia will lead to the development of novel therapeutic strategies.

Connexins: a myriad of functions extending beyond assembly of gap junction channels.

Connexins constitute a large family of trans-membrane proteins that allow intercellular communication and the transfer of ions and small signaling molecules between cells. Recent studies have revealed complex translational and post-translational mechanisms that regulate connexin synthesis, maturation, membrane transport and degradation that in turn modulate gap junction intercellular communication. With the growing myriad of connexin interacting proteins, including cytoskeletal elements, junctional proteins, and enzymes, gap junctions are now perceived, not only as channels between neighboring cells, but as signaling complexes that regulate cell function and transformation. Connexins have also been shown to form functional hemichannels and have roles altogether independent of channel functions, where they exert their effects on proliferation and other aspects of life and death of the cell through mostly-undefined mechanisms. This review provides an updated overview of current knowledge of connexins and their interacting proteins, and it describes connexin modulation in disease and tumorigenesis.

Heterocellular interaction enhances recruitment of alpha and beta-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells.

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation (beta-casein expression) was evaluated. Heterocellular interaction is critical for beta-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complex components (alpha-catenin, beta-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although beta-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and beta-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear beta-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of beta-catenin in GJ complexes.

Anti-inflammatory properties of Salograviolide A purified from Lebanese plant Centaurea ainetensis.

BACKGROUND: Anti-inflammatory activities of medicinal plants have largely been attributed to their content of sesquiterpene lactones (SLs). SLs are predominantly found in the sunflower family Asteraceae and have been isolated from many plants of this family, particularly Centaurea. The anti-inflammatory activities of extract of Centaurea ainetensis, a Lebanese endemic plant, and the isolated active molecule were assessed for their potential ant-inflammatory activities. METHODS: Plant extract from Centaurea ainetensis, and the isolated active ingredient Salograviolide A (SA), a sesquiterpene lactones guaianolide, were used for the study. Western blotting and electrophoretic mobility shift assays were used to test the effects of the plant extract and SA on interleukin-1 (IL-1) induced increase in cyclooxygenase-2 (COX-2) levels and in nuclear factor-kappaB (NF-kappaB) translocation in an intestinal epithelial cell (IEC) of inflammation. Their effects on inflammation score and cytokine levels were also studied in an iodoacetoamide-induced rat model of inflammation. RESULTS: Plant extract and SA were shown to reverse the effects observed by IL-1 on COX-2 levels and NF-kappaB translocation in IEC. SA decreased the level of inflammatory cytokines and the level of inflammation in the animal model. CONCLUSION: These findings suggest that SA may be useful in the development of natural therapies for inflammatory diseases.

Interleukin-6 and Cyclooxygenase-2 downregulation by fatty-acid fractions of Ranunculus constantinopolitanus.

BACKGROUND: Medicinal plants represent alternative means for the treatment of several chronic diseases, including inflammation. The genus Ranunculus, a representative of the Ranunculaceae family, has been reported to possess anti-inflammatory, analgesic, antiviral, antibacterial, antiparasitic and antifungal activities, possibly due to the presence of anemonin and other. Different studies have shown the occurrence of unusual fatty acids (FAs) in Ranunculaceae; however, their therapeutic role has not been investigated. The purpose of this study is to characterize potential anti-inflammatory bioactivities in Ranunculus constantinopolitanus D'Urv., traditionally used in Eastern Mediterranean folk medicine. METHODS: The aerial part of R. constantinopolitanus was subjected to methanol (MeOH) extraction and solvent fractionation. The bioactive fraction (I.2) was further fractionated using column chromatography, and the biologically active subfraction (Y2+3) was identified using infrared (IR) spectroscopy, nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The effects of I.2 and Y2+3 on cell viability were studied in mouse mammary epithelial SCp2 cells using trypan blue exclusion method. To study the anti-inflammatory activities of I.2 and Y2+3, their ability to reduce interleukin (IL)-6 levels was assessed in endotoxin (ET)-stimulated SCp2 cells using enzyme-linked immunosorbent assay (ELISA). In addition, the ability of Y2+3 to reduce cyclooxygenase (COX)-2 expression was studied in IL-1-treated mouse intestinal epithelial Mode-K cells via western blotting. Data were analyzed by one-way analysis of variance (ANOVA), Student-Newman-Keuls (SNK), Tukey HSD, two-sample t-test and Dunnett t-tests for multiple comparisons. RESULTS: The chloroform fraction (I.2) derived from crude MeOH extract of the plant, in addition to Y2+3, a FA mix isolated from this fraction and containing palmitic acid, C18:2 and C18:1 isomers and stearic acid (1:5:8:1 ratio), reduced ET-induced IL-6 levels in SCp2 cells without affecting cell viability or morphology. When compared to fish oil, conjugated linoleic acid (CLA) and to individual FAs as palmitic, linoleic, oleic and stearic acid or to a mix of these FAs (1:5:8:1 ratio), Y2+3 exhibited higher potency in reducing ET-induced IL-6 levels within a shorter period of time. Y2+3 also reduced COX-2 expression in IL-1-treated Mode-K cells. CONCLUSION: Our studies demonstrate the existence of potential anti-inflammatory bioactivities in R. constantinopolitanus and attribute them to a FA mix in this plant.

Connexin43 as a Tumor Suppressor: Proposed Connexin43 mRNA-circularRNAs-microRNAs Axis Towards Prevention and Early Detection in Breast Cancer.

Breast cancer (BC) is a global public health burden, constituting the highest cancer incidence in women worldwide. Connexin43 (Cx43) is a member of a family of transmembrane proteins responsible in part for intercellular communication between adjacent breast epithelial cells, via gap junctions. Cx43 plays key role in mammary gland development and differentiation and its spatio-temporal perturbation contributes to tumorigenesis. Thus, Cx43 acts as a breast tumor-suppressor. Signaling pathways and phenotypes downstream of Cx43 mRNA loss/mis-localization in breast cells have been well-studied. However, axes parallel to Cx43 loss are less understood. microRNAs (miRNAs) are small endogenous non-coding RNAs that repress translation and circularRNAs (circRNAs) are a class of endogenous RNAs that originate from RNA splicing and act as miRNA "sponges". CircRNAs and miRNAs are dysregulated in cancers and are highly abundant and stable in the circulation. Thus, they present as attractive liquid biopsy cancer biomarkers. Here, an axis for Cx43 mRNA-circRNAs-miRNAs interactions along BC initiation (denoted by loss of breast epithelial polarity and development of hyperplastic phenotypes) is proposed to potentially serve as a signature biomarker toward BC early-onset detection and prevention.

Connexin 43 Loss Triggers Cell Cycle Entry and Invasion in Non-Neoplastic Breast Epithelium: A Role for Noncanonical Wnt Signaling.

(1) Background: The expression of connexin 43 (Cx43) is disrupted in breast cancer, and re-expression of this protein in human breast cancer cell lines leads to decreased proliferation and invasiveness, suggesting a tumor suppressive role. This study aims to investigate the role of Cx43 in proliferation and invasion starting from non-neoplastic breast epithelium. (2) Methods: Nontumorigenic human mammary epithelial HMT-3522 S1 cells and Cx43 shRNA-transfected counterparts were cultured under 2-dimensional (2-D) and 3-D conditions. (3) Results: Silencing Cx43 induced mislocalization of ß-catenin and Scrib from apicolateral membrane domains in glandular structures or acini formed in 3-D culture, suggesting the loss of apical polarity. Cell cycle entry and proliferation were enhanced, concomitantly with c-Myc and cyclin D1 upregulation, while no detectable activation of Wnt/ß-catenin signaling was observed. Motility and invasion were also triggered and were associated with altered acinar morphology and activation of ERK1/2 and Rho GTPase signaling, which acts downstream of the noncanonical Wnt pathway. The invasion of Cx43-shRNA S1 cells was observed only under permissive stiffness of the extracellular matrix (ECM). (4) Conclusion: Our results suggest that Cx43 controls proliferation and invasion in the normal mammary epithelium in part by regulating noncanonical Wnt signaling.

A thiosemicarbazone derivative induces triple negative breast cancer cell apoptosis: possible role of miRNA-125a-5p and miRNA-181a-5p.

BACKGROUND: Breast cancer, the most commonly diagnosed malignancy in women, accounts for the highest cancer-related deaths worldwide. Triple negative breast cancer (TNBC), lacking the expression of estrogen, progesterone and HER2 receptors, has an aggressive clinical phenotype and is susceptible to chemotherapy but not to hormonal or targeted immunotherapy. In an attempt to identify potent and selective anti-TNBC agents, a set of thiosemicarbazone derivatives were screened for their cytotoxic activity against MDA-MB 231 breast cancer cell line. METHODS: MTT assay was used to examine cell viability. P53 phosphorylation status, poly (ADP-ribose) polymerase (PARP) cleavage as well as Bcl2 and Bax protein levels were assessed by Western blot. Quantitative Real Time-PCR was carried out to characterize miRNAs expression levels. RESULTS: Combining Cisplatin + thiosemicarbazone compound 4 showed potent anti-TNBC potential. Cisplatin + compound 4 significantly enhanced p53 phosphorylation, induced Bax amount, reduced Bcl2 protein levels, enhanced PARP cleavage and modulated miRNAs expression profile in TNBCs, with a particular overexpression of miR-125a-5p and miR-181a-5p. Intriguingly, miR-125a-5p and miR-181a-5p could significantly downregulate BCL2 expression by binding to their target sites in the 3'UTR. CONCLUSIONS: Collectively, our results demonstrate an anti-TNBC activity of Cisplatin + thiosemicarbazone compound 4 combination mediated via induction of apoptosis.

Cell Culture and Coculture for Oncological Research in Appropriate Microenvironments.

With the increase in knowledge on the importance of the tumor microenvironment, cell culture models of cancers can be adapted to better recapitulate physiologically relevant situations. Three main microenvironmental factors influence tumor phenotype: the biochemical components that stimulate cells, the fibrous molecules that influence the stiffness of the extracellular matrix, and noncancerous cells like epithelial cells, fibroblasts, endothelial cells, and immune cells. Here we present methods for the culture of carcinomas in the presence of a matrix of specific stiffness, and for the coculture of tumors and fibroblasts as well as epithelial cells in the presence of matrix. Information is provided to help with choice and assessment of the matrix support and in working with serum-free medium. Using the example of a tissue chip recapitulating the environmental geometry of carcinomas, we also highlight the development of engineered platforms that provide exquisite control of cell culture parameters necessary in research and development. © 2019 by John Wiley & Sons, Inc.