miR-503-3p promotes epithelialemesenchymal transition in breast cancer by directly targeting SMAD2 and E-cadherin

Although progress in clinical and basic research has significantly increased our understanding of breast cancer, little is known about the molecular mechanism underlying breast cancer metastasis. Identification of effective therapeutic targets to prevent breast cancer metastasis is urgently needed. The function of mi R-503-3p has been investigated in other cancers, but its role in breast cancer remains undefined.Here, we found that mi R-503-3p was overexpressed in breast cancer tissue and plasma compared with adjacent normal breast tissue and with plasma from healthy individuals. Moreover, we identified mi R-503-3p to be an oncogene of breast cancer cell proliferation, migration and invasion. Upregulation of mi R-503-3p in breast cancer cells inhibited expression of epithelialemesenchymal transition(EMT)-related protein SMAD2 and the epithelial marker protein E-cadherin by directly binding to their m RNA30 untranslated region, whereas increased expression of mesenchymal marker proteins, including vimentin and N-cadherin. Taken together, our findings support a critical role for mi R-503-3p in induction of breast cancer EMT and suggest that plasma mi R-503-3p may be a useful diagnostic biomarker for breast cancer.     

Towards a new “stromal-based” classification system for human breast cancer prognosis and therapy

Here, we discuss recent evidence that an absence of stromal Cav-1 expression in human breast cancers is a powerful single independent predictor of early disease recurrence, metastasis, and poor clinical outcome. These findings have now been validated in two independent patient populations. Importantly, the predictive value of stromal Cav-1 is independent of epithelial marker status, making stromal Cav-1 a new “universal” or “widely-applicable” breast cancer prognostic marker. We propose based on the expression of stromal Cav-1, that breast cancer patients could be stratified into high-risk and low-risk groups. High-risk patients showing an absence of stromal Cav-1 should be offered more aggressive therapies, such as anti-angiogenic approaches, in addition to the standard therapy regimens. Mechanistically, loss of stromal Cav-1 is a surrogate biomarker for increased cell cycle progression, growth factor secretion, “stemness”, and angiogenic potential in the tumor microenvironment. Since almost all cancers develop within the context of a stromal microenvironment, this new stromal classification system may be broadly applicable to other epithelial and non-epithelial cancer subtypes, as well as “pre-malignant” lesions (carcinoma in situ).     

Epithelial cell adhesion molecule promotes breast cancer resistance protein-mediated multidrug resistance in breast cancer by inducing partial epithelial–mesenchymal transition

Overexpression of breast cancer resistance protein (BCRP) plays a crucial role in the acquired multidrug resistance (MDR) in breast cancer. The elucidation of molecular events that confer BCRP-mediated MDR is of major therapeutic importance in breast cancer. Epithelial cell adhesion molecule (EpCAM) has been implicated in tumor progression and drug resistance in various types of cancers, including breast cancer. However, the role of EpCAM in BCRP-mediated MDR in breast cancer remains unknown. In the present study, we revealed that EpCAM expression was upregulated in BCRP-overexpressing breast cancer MCF-7/MX cells, and EpCAM knockdown using siRNA reduced BCRP expression and increased the sensitivity of MCF-7/MX cells to mitoxantrone (MX). The epithelial–mesenchymal transition (EMT) promoted BCRP-mediated MDR in breast cancer cells, and EpCAM knockdown partially suppressed EMT progression in MCF-7/MX cells. In addition, Wnt/β-catenin signaling was activated in MCF-7/MX cells, and the inhibition of this signaling attenuated EpCAM and BCRP expression and partially reversed EMT. Together, this study illustrates that EpCAM upregulation by Wnt/β-catenin signaling induces partial EMT to promote BCRP-mediated MDR resistance in breast cancer cells. EpCAM may be a potential therapeutic target for overcoming BCRP-mediated resistance in human breast cancer.     

The multifaceted roles of neutrophil gelatinase associated lipocalin (NGAL) in inflammation and cancer

Neutrophil gelatinase associated lipocalin (NGAL), also known as oncogene 24p3, uterocalin, siderocalin or lipocalin 2, is a 24kDa secreted glycoprotein originally purified from a culture of mouse kidney cells infected with simian virus 40 (SV-40). Subsequent investigations have revealed that it is a member of the lipocalin family of proteins that transport small, hydrophobic ligands. Since then, NGAL expression has been reported in several normal tissues where it serves to provide protection against bacterial infection and modulate oxidative stress. Its expression is also dysregulated in several benign and malignant diseases. Its small size, secreted nature and relative stability have led to it being investigated as a diagnostic and prognostic biomarker in numerous diseases including inflammation and cancer. Functional studies, conducted primarily on lipocalin 2 (Lcn2), the mouse homologue of human NGAL have revealed that Lcn2 has a strong affinity for iron complexed to both bacterial siderophores (iron-binding proteins) and certain human proteins like norepinephrine. By sequestering iron-laden siderophores, Lcn2 deprives bacteria of a vital nutrient and thus inhibits their growth (bacteriostatic effect). In malignant cells, its proposed functions range from inhibiting apoptosis (in thyroid cancer cells), invasion and angiogenesis (in pancreatic cancer) to increasing proliferation and metastasis (in breast and colon cancer). Ectopic expression of Lcn2 also promotes BCR-ABL induced chronic myelogenous leukemia in murine models. By transporting iron into and out of the cell, NGAL also regulates iron responsive genes. Further, it stabilizes the proteolytic enzyme matrix metalloprotease-9 (MMP-9) by forming a complex with it, and thereby prevents its autodegradation. The factors regulating NGAL expression are numerous and range from pro-inflammatory cytokines like interleukins, tumor necrosis factor-α and interferons to vitamins like retinoic acid. The purpose of this review article is to examine the expression, structure, regulation and biological role of NGAL and critically assess its potential as a novel diagnostic and prognostic marker in both benign and malignant human diseases.     

Mitotic kinases as drivers of the epithelial-to-mesenchymal transition and as therapeutic targets against breast cancers

Biological therapies against breast cancer patients with tumors positive for the estrogen and progesterone hormone receptors and Her2 amplification have greatly improved their survival. However, to date, there are no effective biological therapies against breast cancers that lack these three receptors or triple-negative breast cancers (TNBC). TNBC correlates with poor survival, in part because they relapse following chemo- and radio-therapies. TNBC is intrinsically aggressive since they have high mitotic indexes and tend to metastasize to the central nervous system. TNBCs are more likely to display centrosome amplification, an abnormal phenotype that results in defective mitotic spindles and abnormal cytokinesis, which culminate in aneuploidy and chromosome instability (known causes of tumor initiation and chemo-resistance). Besides their known role in cell cycle control, mitotic kinases have been also studied in different types of cancer including breast, especially in the context of epithelial-to-mesenchymal transition (EMT). EMT is a cellular process characterized by the loss of cell polarity, reorganization of the cytoskeleton, and signaling reprogramming (upregulation of mesenchymal genes and downregulation of epithelial genes). Previously, we and others have shown the effects of mitotic kinases like Nek2 and Mps1 (TTK) on EMT. In this review, we focus on Aurora A, Aurora B, Bub1, and highly expressed in cancer (Hec1) as novel targets for therapeutic interventions in breast cancer and their effects on EMT. We highlight the established relationships and interactions of these and other mitotic kinases, clinical trial studies involving mitotic kinases, and the importance that represents to develop drugs against these proteins as potential targets in the primary care therapy for TNBC.     

E-cadherin's dark side: Possible role in tumor progression

In the context of cancer, E-cadherin has traditionally been categorized as a tumor suppressor, given its essential role in the formation of proper intercellular junctions, and its downregulation in the process of epithelial-mesenchymal transition (EMT) in epithelial tumor progression. Germline or somatic mutations in the E-cadherin gene (CDH1) or downregulation by epigenetic mechanisms have been described in a small subset of epithelial cancers. However, recent evidence also points toward a promoting role of E-cadherin in several aspects of tumor progression. This includes preserved (or increased) E-cadherin expression in microemboli of inflammatory breast carcinoma, a possible "mesenchymal to epithelial transition" (MET) in ovarian carcinoma, collective cell invasion in some epithelial cancers, a recent association of E-cadherin expression with a more aggressive brain tumor subset, as well as the intriguing possibility of E-cadherin involvement in specific signaling networks in the cytoplasm and/or nucleus. In this review we address a lesser-known, positive role for E-cadherin in cancer.     

Lin28 Induces Epithelial-to-Mesenchymal Transition and Stemness via Downregulation of Let-7a in Breast Cancer Cells

The RNA-binding protein Lin28 is known to promote malignancy by inhibiting the biogenesis of let-7, which functions as a tumor suppressor. However, the role of the Lin28/let-7 axis in the epithelial-to-mesenchymal transition (EMT) and stemness in breast cancer has not been clearly expatiated. In our previous study, we demonstrated that let-7 regulates self-renewal and tumorigenicity of breast cancer stem cells. In the present study, we demonstrated that Lin28 was highly expressed in mesenchymal (M) type cells (MDA-MB-231 and SK-3rd), but it was barely detectable in epithelial (E) type cells (MCF-7 and BT-474). Lin28 remarkably induced the EMT, increased a higher mammosphere formation rate and ALDH activity and subsequently promoted colony formation, as well as adhesion and migration in breast cancer cells. Furthermore, we demonstrated that Lin28 induced EMT in breast cancer cells via downregulation of let-7a. Strikingly, Lin28 overexpression was found in breast cancers that had undergone metastasis and was strongly predictive of poor prognoses in breast cancers. Given that Lin28 induced the EMT via let-7a and promoted breast cancer metastasis, Lin28 may be a therapeutic target for the eradication of breast cancer metastasis.     

Breast cancer cells: Focus on the consequences of epithelial-to-mesenchymal transition

Breast cancers are highly heterogeneous and successful treatment of those subtypes with a high frequency of metastases and resistance to clinically available therapies remains a challenge. An understanding of mechanisms which may contribute to this heterogeneity and generation of more resilient cancer cells is therefore essential. Epithelial-to-mesenchymal transition (EMT) is a dynamic two-way process that occurs during embryonic development and wound healing whereby epithelial cells can gain plasticity and switch to a mesenchymal-like phenotype. EMT has received interest from cancer researchers due to its potential role in processes important in cancer progression and metastasis. Recent evidence has revealed a clear association between EMT and resistance to therapeutics. Targeting of EMT and/or the mesenchymal-like phenotype may be a promising avenue for future therapeutic intervention. This review provides a brief summary of the functional consequences of EMT in breast cancer, with a focus on the mesenchymal-like phenotype.     

E-cadherin's dark side: Possible role in tumor progression

In the context of cancer, E-cadherin has traditionally been categorized as a tumor suppressor, given its essential role in the formation of proper intercellular junctions, and its downregulation in the process of epithelial–mesenchymal transition (EMT) in epithelial tumor progression. Germline or somatic mutations in the E-cadherin gene (CDH1) or downregulation by epigenetic mechanisms have been described in a small subset of epithelial cancers. However, recent evidence also points toward a promoting role of E-cadherin in several aspects of tumor progression. This includes preserved (or increased) E-cadherin expression in microemboli of inflammatory breast carcinoma, a possible “mesenchymal to epithelial transition” (MET) in ovarian carcinoma, collective cell invasion in some epithelial cancers, a recent association of E-cadherin expression with a more aggressive brain tumor subset, as well as the intriguing possibility of E-cadherin involvement in specific signaling networks in the cytoplasm and/or nucleus. In this review we address a lesser-known, positive role for E-cadherin in cancer.     

Derivation of myoepithelial progenitor cells from bipotent mammary stem/progenitor cells

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Recent molecular profiling has identified six major subtypes of breast cancer: basal-like, ErbB2-overexpressing, normal breast epithelial-like, luminal A and B, and claudin-low subtypes. To help understand the relationship among mammary stem/progenitor cells and breast cancer subtypes, we have recently derived distinct hTERT-immortalized human mammary stem/progenitor cell lines: a K5(+)/K19(-) type, and a K5(+)/K19(+) type. Under specific culture conditions, bipotent K5(+)/K19(-) stem/progenitor cells differentiated into stable clonal populations that were K5(-)/K19(-) and exhibit self-renewal and unipotent myoepithelial differentiation potential in contrast to the parental K5(+)/K19(-) cells which are bipotent. These K5(-)/K19(-) cells function as myoepithelial progenitor cells and constitutively express markers of an epithelial to mesenchymal transition (EMT) and show high invasive and migratory abilities. In addition, these cells express a microarray signature of claudin-low breast cancers. The EMT characteristics of an un-transformed unipotent mammary myoepithelial progenitor cells together with claudin-low signature suggests that the claudin-low breast cancer subtype may arise from myoepithelial lineage committed progenitors. Availability of immortal MPCs should allow a more definitive analysis of their potential to give rise to claudin-low breast cancer subtype and facilitate biological and molecular/biochemical studies of this disease.     

Diverse and converging roles of ERK1/2 and ERK5 pathways on mesenchymal to epithelial transition in breast cancer

The epithelial to mesenchymal transition (EMT) is characterized by a loss of cell polarity, a decrease in the epithelial cell marker E-cadherin, and an increase in mesenchymal markers including the zinc-finger E-box binding homeobox (ZEB1). The EMT is also associated with an increase in cell migration and anchorage-independent growth. Induction of a reversal of the EMT, a mesenchymal to epithelial transition (MET), is an emerging strategy being explored to attenuate the metastatic potential of aggressive cancer types, such as triple-negative breast cancers (TNBCs) and tamoxifen-resistant (TAMR) ER-positive breast cancers, which have a mesenchymal phenotype. Patients with these aggressive cancers have poor prognoses, quick relapse, and resistance to most chemotherapeutic drugs. Overexpression of extracellular signal-regulated kinase (ERK) 1/2 and ERK5 is associated with poor patient survival in breast cancer. Moreover, TNBC and tamoxifen resistant cancers are unresponsive to most targeted clinical therapies and there is a dire need for alternative therapies.In the current study, we found that MAPK3, MAPK1, and MAPK7 gene expression correlated with EMT markers and poor overall survival in breast cancer patients using publicly available datasets. The effect of ERK1/2 and ERK5 pathway inhibition on MET was evaluated in MDA-MB-231, BT-549 TNBC cells, and tamoxifen-resistant MCF-7 breast cancer cells. Moreover, TU-BcX-4IC patient-derived primary TNBC cells were included to enhance the translational relevance of our study. We evaluated the effect of pharmacological inhibitors and lentivirus-induced activation or inhibition of the MEK1/2-ERK1/2 and MEK5-ERK5 pathways on cell morphology, E-cadherin, vimentin and ZEB1 expression. Additionally, the effects of pharmacological inhibition of trametinib and XMD8-92 on nuclear localization of ERK1/2 and ERK5, cell migration, proliferation, and spheroid formation were evaluated. Novel compounds that target the MEK1/2 and MEK5 pathways were used in combination with the AKT inhibitor ipatasertib to understand cell-specific responses to kinase inhibition. The results from this study will aid in the design of innovative therapeutic strategies that target cancer metastases.     

Downregulation of CFTR promotes epithelial-to-mesenchymal transition and is associated with poor prognosis of breast cancer

The epithelial-to-mesenchymal transition (EMT), a process involving the breakdown of cell–cell junctions and loss of epithelial polarity, is closely related to cancer development and metastatic progression. While the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl^? and HCO₃^? conducting anion channel expressed in a wide variety of epithelial cells, has been implicated in the regulation of epithelial polarity, the exact role of CFTR in the pathogenesis of cancer and its possible involvement in EMT process have not been elucidated. Here we report that interfering with CFTR function either by its specific inhibitor or lentiviral miRNA-mediated knockdown mimics TGF-β₁-induced EMT and enhances cell migration and invasion in MCF-7. Ectopic overexpression of CFTR in a highly metastatic MDA-231 breast cancer cell line downregulates EMT markers and suppresses cell invasion and migration in vitro, as well as metastasis in vivo. The EMT-suppressing effect of CFTR is found to be associated with its ability to inhibit NFκB targeting urokinase-type plasminogen activator (uPA), known to be involved in the regulation of EMT. More importantly, CFTR expression is found significantly downregulated in primary human breast cancer samples, and is closely associated with poor prognosis in different cohorts of breast cancer patients. Taken together, the present study has demonstrated a previously undefined role of CFTR as an EMT suppressor and its potential as a prognostic indicator in breast cancer.     

Estrogen promotes reversible epithelial-to-mesenchymal-like transition and collective motility in MCF-7 breast cancer cells

The role of estrogen in the motility and invasion of breast cancer cells is controversial. Although estrogen receptor (ER)-positive breast tumors are considered less aggressive and more differentiated they still undergo metastasis. In many types of epithelial cancers, the ability to undergo metastasis has been associated with a loss of epithelial features and acquisition of mesenchymal properties leading to migration of individual cells, a process known as epithelial-to-mesenchymal transition (EMT). In this report, we show that a subset of ER-positive breast cancer cells can acquire mesenchymal-like features and motility in a reversible manner. In MCF-7 breast cancer cells estrogen-promoted acquisition of mesenchymal-like features while antiestrogens, such as tamoxifen, prevented this transition. Moreover, pharmacological inhibition of Src family kinases decreased the ability of estrogen to promote epithelial-to-mesenchymal-like transition. In addition to mesenchymal-like motility, a subset of estrogen-treated cells also moved as cell clusters (collective motility). While membrane localization of E-cadherin/beta-catenin was decreased in fibroblast-like cells, enhanced levels of E-cadherin/beta-catenin were detected in motile cell clusters. Thus, during tumor progression, estrogen may foster motility and invasion of ER-positive breast cancer by promoting simultaneously reversible EMT-like changes and collective motility. These studies suggest that antiestrogen therapy and Src family kinase inhibitors may decrease development of metastases in ER-positive breast cancer by blocking estrogen-dependent migration of human breast cancer cells.