MALAT-1: Immunomodulatory lncRNA hampering the innate and the adaptive immune arms in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) is known as hot immunogenic tumor. Yet, it is one of the most aggressive BC subtypes. TNBC evolve several tactics to evade the immune surveillance phenomena, one of which is shedding of natural killer (NK) cells activating immune ligands such as MICA/B and/or by inducing the expression of the immune checkpoints such as PD-L1 and B7-H4. MALAT-1 is an oncogenic lncRNA. MALAT-1 immunogenic profile is not well investigated.AimThe study aims at exploring the immunogenic role of MALAT-1 in TNBC patients and cell lines and to identify its molecular mechanism in altering both innate and adaptive immune cells present at the tumor microenvironment of TNBCMethodsBC patients (n = 35) were recruited. Primary NK cells and cytotoxic T lymphocytes were isolated from normal individuals using the negative selection method. MDA-MB-231 cells were cultured and transfected by several oligonucleotides by lipofection technique. Screening of ncRNAs was performed using q-RT-PCR. Immunological functional analysis experiments were performed upon co-culturing primary natural killer cells and cytotoxic T lymphocytes using LDH assay. Bioinformatics analysis was performed to identify potential microRNAs targeted by MALAT-1.ResultsMALAT-1 expression was significantly upregulated in BC patinets with a profound expression in TNBC patients compared to their normal counterparts. Correlation analysis revealed a positive correlation between MALAT-1, tumor size and lymph node metastasis. Knocking down of MALAT-1 in MDA-MB-231 cells resulted in a significant induction of MICA/B, repression of PD-L1 and B7-H4 expression levels. Enhancement of cytotoxic activity of co-cultured NK and CD8⁺ cells with MALAT-1 siRNAs transfected MDA-MB-231 cells. In silico analysis revealed that miR-34a and miR-17–5p are potential targets to MALAT-1; accordingly, they were found to be downregulated in BC patients. Forcing the expression of miR-34a in MDA-MB-231 cells resulted in a significant induction in MICA/B levels. Ectopic expression of miR-17–5p in MDA-MB-231 cells significantly repressed the expression of PD-L1 and B7-H4 checkpoints. Validations of MALAT-1/miR-34a" and "MALAT-1/miR-17–5p axes were performed by a series of co-transfections and functional assessment of cytotoxic profile of primary immune cells.ConclusionThis study proposes a novel epigenetic alteration exerted by TNBC cells mainly by inducing the expression of MALAT-1 lncRNA. MALAT-1 mediates innate and adaptive immune suppression events partially via targeting miR-34a/MICA/B and miR-175p/PD-L1/B7-H4 axes in TNBC patients and cell lines.     

Pharmacological inhibition of the MEK5/ERK5 and PI3K/Akt signaling pathways synergistically reduces viability in triple-negative breast cancer

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.     

The lysosomal TRPML1 channel regulates triple negative breast cancer development by promoting mTORC1 and purinergic signaling pathways

The triple-negative breast cancer (TNBC) that comprises approximately 10%–20% of breast cancers is an aggressive subtype lacking effective therapeutics. Among various signaling pathways, mTORC1 and purinergic signals have emerged as potentially fruitful targets for clinical therapy of TNBC. Unfortunately, drugs targeting these signaling pathways do not successfully inhibit the progression of TNBC, partially due to the fact that these signaling pathways are essential for the function of all types of cells. In this study, we report that TRPML1 is specifically upregulated in TNBCs and that its genetic downregulation and pharmacological inhibition suppress the growth of TNBC. Mechanistically, we demonstrate that TRPML1 regulates TNBC development, at least partially, through controlling mTORC1 activity and the release of lysosomal ATP. Because TRPML1 is specifically activated by cellular stresses found in tumor microenvironments, antagonists of TRPML1 could represent anticancer drugs with enhanced specificity and potency. Our findings are expected to have a major impact on drug targeting of TNBCs.     

Clinicopathological and biological significance of human voltage-gated proton channel Hv1 protein overexpression in breast cancer

In our previous work, we showed for the first time that the voltage-gated proton channel Hv1 is specifically expressed in highly metastatic human breast tumor tissues and cell lines. However, the contribution of Hv1 to breast carcinogenesis is not well known. In this study, we showed that Hv1 expression was significantly correlated with the tumor size (p = 0.001), tumor classification (p = 0.000), lymph node status (p = 0.000), clinical stage (p = 0.000), and Her-2 status (p = 0.045). High Hv1 expression was associated significantly with shorter overall (p = 0.000) and recurrence-free survival (p = 0.000). In vitro, knockdown of Hv1 expression in the highly metastatic MDA-MB-231 cells decreased the cell proliferation and invasiveness, inhibited the cell proton secretion and intracellular pH recovery, and blocked the cell capacity of acidifying extracellular milieu. Furthermore, the gelatinase activity in MDA-MB-231 cells that suppressed Hv1 was reduced. In vivo, the breast tumor size of the implantation of the MDA-MB-231 xenografts in nude mice that were knocked down by Hv1 was dramatically smaller than that in the control groups. The results demonstrated that the inhibition of Hv1 function via knockdown of Hv1 expression can effectively retard the cancer growth and suppress the cancer metastasis by the decrease of proton extrusion and the down-regulation of gelatinase activity. Based on these results, we came to the conclusion that Hv1 is a potential biomarker for prognosis of breast cancer and a potential target for anticancer drugs in breast cancer therapy.     

HIF1A-AS2 predicts poor prognosis and regulates cell migration and invasion in triple-negative breast cancer

The aberrant expression of hypoxia-inducible factor 1 alpha (HIF1A)-antisense RNA 2 (HIF1A-AS2) was found in various human cancers including breast cancer. The aim of this study was to present more evidence about the role HIF1A-AS2 on triple-negative breast cancer (TNBC). In our results, HIF1A-AS2 was also found to be upregulated in TNBC tissues compared with non-TNBC tissues or adjacent normal tissues. Besides, HIF1A-AS2 expression was also elevated in TNBC cell lines compared with the normal breast epithelial cell line. Moreover, high expression of HIF1A-AS2 was associated with lymph node metastasis, distant metastasis and unfavorable histological grade in TNBC patients. Survival analysis showed a TNBC patient with high HIF1A-AS2 expression had shorter overall survival than patients with low HIF1A-AS2 expression, and HIF1A-AS2 high expression acted as an independent poor prognostic factor for overall survival in TNBC patients. The cell migration and invasion assays suggested inhibition of HIF1A-AS2 obviously depressed TNBC cell migration and invasion. In conclusion, HIF1A-AS2 serves as a novel biomarker for predicting clinical progression and prognosis in TNBC.     

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.     

Lamin B2 promotes the progression of triple negative breast cancer via mediating cell proliferation and apoptosis

Triple negative breast cancer (TNBC) is a more common type of breast cancer with high distant metastasis and poor prognosis. The potential role of lamins in cancer progression has been widely revealed. However, the function of lamin B2 (LMNB2) in TNBC progression is still unclear. The present study aimed to investigate the role of LMNB2 in TNBC. The cancer genome atlas (TCGA) database analysis and immunohistochemistry (IHC) were performed to examine LMNB2 expression levels. LMNB2 short hairpin RNA plasmid or lentivirus was used to deplete the expression of LMNB2 in human TNBC cell lines including MDA-MB-468 and MDA-MB-231. Alterations in cell proliferation and apoptosis in vitro and the nude mouse tumorigenicity assay in vivo were subsequently analyzed. The human TNBC tissues shown high expression of LMNB2 according to the bioinformation analysis and IHC assays. LMNB2 expression was correlated with the clinical pathological features of TNBC patients, including pTNM stage and lymph node metastasis. Through in vitro and in vivo assays, we confirmed LMNB2 depletion suppressed the proliferation and induced the apoptosis of TNBC cells, and inhibited tumor growth of TNBC cells in mice, with the decrease in Ki67 expression or the increase in caspase-3 expression. In conclusion, LMNB2 may promote TNBC progression and could serve as a potential therapeutic target for TNBC treatment.     

SENP1 promotes triple-negative breast cancer invasion and metastasis via enhancing CSN5 transcription mediated by GATA1 deSUMOylation

TNBC is characterized by high incidence of visceral metastasis and lacks effective clinical targets. This study aims to delineate the molecular mechanisms of SENP1 in TNBC invasion and metastasis. By using IHC to test the SENP1 expression in TNBC tissues, we analyzed the relationship between SENP1 expression and TNBC prognosis. We showed that SENP1 expression was higher in TNBC tumor tissues and related to TNBC prognosis, supporting SENP1 as an independent risk factor. High expression of SENP1 was significantly associated with histologic grade and tumor lymph node invasion. Intriguingly, the expression levels of SENP1 in TNBC tumors were significantly correlated with that of CSN5, GATA1 and ZEB1. Importantly, SENP1 promoted TNBC cell migration and invasion by regulating ZEB1 deubiquitination and expression through CSN5. Further studies showed that deSUMOylation at lysine residue K137 of GATA1 enhanced the binding of GATA1 to the CSN5 promoter and transactivated CSN5 expression. In addition, we showed that ZEB1 is deubiquitinated at lysine residue K1108. Our in vivo studies also indicated that reduction in SENP1 expression upregulated GATA1 SUMOylation, and thus resulted in decreased expression of CSN5 and ZEB1 in the tumor microenvironment, which decelerated TNBC progression and metastasis. SENP1 promoted CSN5-mediated ZEB1 protein degradation via deSUMOylation of GATA1, and thus influenced TNBC progression. These findings suggest that SENP1 could be utilized as a potential target for blockade of TNBC development and thus provide a totally new approach for TNBC treatment.     

NHE1 mediates MDA-MB-231 cells invasion through the regulation of MT1-MMP

Na⁺/H⁺ exchanger 1 (NHE1), an important regulator of intracellular pH (pH_i) and extracellular pH (pH_e), has been shown to play a key role in breast cancer metastasis. However, the exact mechanism by which NHE1 mediates breast cancer metastasis is not yet well known. We showed here that inhibition of NHE1 activity, with specific inhibitor Cariporide, could suppress MDA-MB-231 cells invasion as well as the activity and expression of MT1-MMP. Overexpression of MT1-MMP resulted in a distinguished increase in MDA-MB-231 cells invasiveness, but treatment with Cariporide reversed the MT1-MMP-mediated enhanced invasiveness. To explore the role of MAPK signaling pathways in NHE1-mediated breast cancer metastasis, we compared the difference of constitutively phosphorylated ERK1/2, p38 MAPK and JNK in non-invasive MCF-7 cells and invasive MDA-MB-231cells. Interestingly, we found that the phosphorylation levels of ERK1/2 and p38 MAPK in MDA-MB-231 cells were higher than in MCF-7 cells, but both MCF-7 cells and MDA-MB-231 cells expressed similar constitutively phosphorylated JNK. Treating MDA-MB-231 cells with Cariporide led to decreased phosphorylation level of both p38 MAPK and ERK1/2 in a time-dependent manner, but JNK activity was not influenced. Supplementation with MAPK inhibitor (MEK inhibitor PD98059, p38 MAPK inhibitor SB203580 and JNK inhibitor SP600125) or Cariporide all exhibited significant depression of MDA-MB-231 cells invasion and MT1-MMP expression. Furthermore, we co-treated MDA-MB-231 cells with MAPK inhibitor and Cariporide. The result showed that Cariporide synergistically suppressed invasion and MT1-MMP expression with MEK inhibitor and p38 MAPK inhibitor, but not be synergistic with the JNK inhibitor. These findings suggest that NHE1 mediates MDA-MB-231 cells invasion partly through regulating MT1-MMP in ERK1/2 and p38 MAPK signaling pathways dependent manner.     

Sulforaphane-cisplatin combination inhibits the stemness and metastatic potential of TNBCs via down regulation of sirtuins-mediated EMT signaling axis

BackgroundSulforaphane (SFN) is a naturally occurring organosulfur compound found in cruciferous vegetables such as broccoli, brussels sprouts and cabbage. SFN is known for its multiple therapeutic properties, such as HDAC inhibitory, chemo preventive and anti-cancer effects. Cisplatin (CIS) has limited effect against metastatic triple-negative breast cancer (TNBC). Additionally, CIS impose severe side effects to normal cells, and later TNBC cells develops resistance. Studies suggest that the overexpression of sirtuins (SIRTs) promotes CIS resistance and metastasis by activating epithelial-to-mesenchymal transition (EMT) pathway in TNBC.PurposeIn view of the above information, we investigated the therapeutic efficacy of SFN, in combination with CIS against TNBC metastasis and CIS resistance.MethodsThe anti-cancerous effect of SFN-CIS combination on human TNBC cell lines was demonstrated by utilizing MTT assay and, apoptosis and cell cycle assay followed by FACS analysis. The synergistic effect of SFN-CIS combination on the experimental metastasis was demonstrated by utilizing migration, invasion, chemotaxis, mammosphere and colony formation assay on human TNBC MDA-MB-231 and MDA-MB-468 cells. The role of SIRTs-mediated EMT signaling axis in the metastasis and chemoresistance was investigated by western blotting technique as well as sirtuin activity tests. This was further validated by using Chromatin immunoprecipitation (ChIP) analysis.ResultsWe found that SFN-CIS combination synergistically inhibits cellular growth of MDA-MB-231 and MDA-MB-468 cells. More importantly, SFN was found to protect normal kidney cells from CIS-induced toxicity. Further, SFN-CIS combination was found to synergistically inhibit metastatic-events via significantly altering EMT markers which was further associated with the suppression of SIRTs functions in TNBC cells. ChIP analysis validated that SFN-CIS combination suppresses EMT mechanism through altered chromatin modifications at E-cadherin promoter resulting in its re-expression.ConclusionThe results of the current study suggests that CIS when supplemented with SFN, inhibits metastasis and stemness potential of TNBC cells by down regulating SIRTs-mediated EMT cascade. Overall this study affirms that, this novel combination could be a promising strategy against SIRT-mediated TNBC metastasis and CIS-resistance.     

An SAR study of hydroxy-trifluoromethylpyrazolines as inhibitors of Orai1-mediated store operated Ca2+ entry in MDA-MB-231 breast cancer cells using a convenient Fluorescence Imaging Plate Reader assay

The proteins Orai1 and STIM1 control store-operated Ca²⁺ entry (SOCE) into cells. SOCE is important for migration, invasion and metastasis of MDA-MB-231 human triple negative breast cancer (TNBC) cells and has been proposed as a target for cancer drug discovery. Two hit compounds from a medium throughput screen, displayed encouraging inhibition of SOCE in MDA-MB-231 cells, as measured by a Fluorescence Imaging Plate Reader (FLIPR) Ca²⁺ assay. Following NMR spectroscopic analysis of these hits and reassignment of their structures as 5-hydroxy-5-trifluoromethylpyrazolines, a series of analogues was prepared via thermal condensation reactions between substituted acylhydrazones and trifluoromethyl 1,3-dicarbonyl arenes. Structure-activity relationship (SAR) studies showed that small lipophilic substituents at the 2- and 3-positions of the RHS and 2-, 3- and 4-postions of the LHS terminal benzene rings improved activity, resulting in a novel class of potent and selective inhibitors of SOCE.     

Tumor-derived exosomal circPSMA1 facilitates the tumorigenesis,metastasis, and migration in triple-negative breast cancer (TNBC) through miR-637/Akt1/β-catenin (cyclin D1) axis

Circular RNAs (circRNAs) are increasingly gaining importance and attention due to their diverse potential functions and their value as diagnostic biomarkers (disease specific). This study aims to explore the novel mechanisms by which exosome-contained circRNAs promote tumor development and metastasis in TNBC. We identified increased circRNA circPSMA1 in TNBC cells, their exosomes, and serum exosomes samples from TNBC patients. The overexpression of circPSMA1 promoted TNBC cell proliferation, migration, and metastasis both in vitro and in vivo. Moreover, we investigated the tumor-infiltrating immune cells (TICs) or stromal components in immune microenvironment (IME), and identified the significant differences in the immune cells between TNBC and non-TNBC samples. Mechanistically, circPSMA1 acted as a “miRNAs sponge” to absorb miR-637; miR-637 inhibited TNBC cell migration and metastasis by directly targeted Akt1, which recognized as a key immune-related gene and affected downstream genes β-catenin and cyclin D1. Subsequent co-culture experiments also demonstrated that exosomes from TNBC carrying large amounts of circPSMA1 could transmit migration and proliferation capacity to recipient cells. Kaplan–Meier plots showed that high expression of Akt1 and low expression of mir-637 are highly correlated with poor prognosis in patients with lymph node metastasis of TNBC. Collectively, all these results reveal that circPSMA1 functions as a tumor promoter through the circPSMA1/miR-637/Akt1-β-catenin (cyclin D1) regulatory axis, which can facilitate the tumorigenesis, metastasis, and immunosuppression of TNBC. Our research proposes a fresh perspective on novel potential biomarkers and immune treatment strategies for TNBC.Subject terms: Breast cancer, Cancer microenvironment     

Annexin A1 interaction with the FPR2/ALX receptor: identification of distinct domains and downstream associated signaling

Understanding how proresolving agonists selectively activate FPR2/ALX is a crucial step in the clarification of proresolution molecular networks that can be harnessed for the design of novel therapeutics for inflammatory disease. FPR2/ALX, a G protein-coupled receptor belonging to the formyl peptide receptor (FPR) family, conveys the biological functions of a variety of ligands, including the proresolution mediators annexin A1 (AnxA1) and lipoxin A(4), as well as the activating and proinflammatory protein serum amyloid A. FPR2/ALX is the focus of intense screening for novel anti-inflammatory therapeutics, and the small molecule compound 43 was identified as a receptor ligand. Here, we used chimeric FPR1 and FPR2/ALX clones (stably transfected in HEK293 cells) to identify the N-terminal region and extracellular loop II as the FPR2/ALX domain required for AnxA1-mediated signaling. Genomic responses were also assessed with domain-specific effects emerging, so the N-terminal region is required for AnxA1 induction of JAG1 and JAM3, whereas it is dispensable for modulation of SGPP2. By comparison, serum amyloid A non-genomic responses were reliant on extracellular loops I and II, whereas the small molecule compound 43 activated extracellular loop I with downstream signaling dependent on transmembrane region II. In desensitization experiments, the N-terminal region was dispensable for AnxA1-induced FPR2/ALX down-regulation in both the homologous and heterologous desensitization modes.     

Increased Expression Levels of WAVE3 Are Associated with the Progression and Metastasis of Triple Negative Breast Cancer

Background

Breast Cancer (BC) is a heterogeneous disease comprised of at least five genetically distinct subtypes, which together form the second leading cause of cancer death in women in the United States. Within BC subtypes, those classified as Triple Negative BCs (TNBCs) exhibit dismal survival rates due to their propensity to develop distant metastases. We have identified the WAVE3 protein, which is a critical regulator of actin cytoskeleton dynamics that are required for the motility and invasion of cancer cells through its activation of the Arp2/3 complex, as a key regulator of the different steps of the invasion-metastasis cascade in BC, especially in the more aggressive TNBCs. Our published studies have also shown that elevated expression levels of WAVE3 in the TNBC cell lines directly contribute to their increased invasion and metastasis potentials both in vitro and in vivo in murine models of BC metastasis.

Methodology/Principal Findings

Herein, we utilized both immunohistochemistry (IHC) of primary human BC tumors as well as quantitative real-time RT-PCR of WAVE3 in the peripheral blood of BC patients to clearly establish that WAVE3 is a predictive marker of overall BC patients’ survival. High levels of WAVE3 were predictive for reduced distant recurrence-free survival as well as for decreased disease-specific mortality. Our analysis of WAVE3 expression levels in the peripheral blood of BC patients showed that WAVE3 is highly expressed in the blood of patients who developed metastatic breast cancer compared to those who did not. WAVE3 expression was also highly upregulated in the blood of BC patients with the more aggressive TNBC subtype.

Conclusions

Together, these findings establish WAVE3 as a novel marker for increased risk of breast-cancer-specific mortality and for the metastatic potential of the TNBCs, and also identify WAVE3 as an attractive therapeutic target for the treatment of metastatic BC.     

MiR-487a Promotes TGF-β1-induced EMT,the Migration and Invasion of Breast Cancer Cells by Directly Targeting MAGI2

Tumor metastasis is a complex and multistep process and its exact molecular mechanisms remain unclear. We attempted to find novel microRNAs (miRNAs) contributing to the migration and invasion of breast cancer cells. In this study, we found that the expression of miR-487a was higher in MDA-MB-231breast cancer cells with high metastasis ability than MCF-7 breast cancer cells with low metastasis ability and the treatment with transforming growth factor β1 (TGF-β1) significantly increased the expression of miR-487a in MCF-7 and MDA-MB-231 breast cancer cells. Subsequently, we found that the transfection of miR-487a inhibitor significantly decreased the expression of vimentin, a mesenchymal marker, while increased the expression of E-cadherin, an epithelial marker, in both MCF-7 cells and MDA-MB-231 cells. Also, the inactivation of miR-487a inhibited the migration and invasion of breast cancer cells. Furthermore, our findings demonstrated that miR-487a directly targeted the MAGI2 involved in the stability of PTEN. The down-regulation of miR-487a increased the expression of p-PTEN and PTEN, and reduced the expression of p-AKT in both cell lines. In addition, the results showed that NF-kappaB (p65) significantly increased the miR-487a promoter activity and expression, and TGF-β1 induced the increased miR-487a promoter activity via p65 in MCF-7 cells and MDA-MB-231 cells. Moreover, we further confirmed the expression of miR-487a was positively correlated with the lymph nodes metastasis and negatively correlated with the expression of MAGI2 in human breast cancer tissues. Overall, our results suggested that miR-487a could promote the TGF-β1-induced EMT, the migration and invasion of breast cancer cells by directly targeting MAGI2.     

Subcellular localization of total and activated Src kinase in African American and Caucasian breast cancer

Background

Src, a non-receptor tyrosine kinase is elevated in cancer with expression and activity correlated with cell proliferation, adhesion, survival, motility, metastasis and angiogenesis. There is limited data on Src expression and subcellular localization in breast cancer and no information about expression in racial/ethnic groups.

Methodology/Principal Findings

The present study evaluated Src expression, activity, and subcellular localization in triple negative breast cancer (TNBC) and ERα positive breast cancer (ER+BC), cancer tissue and adjacent normal epithelial ducts, and Caucasian and African American cases. 79 paraffin embedded breast carcinoma cases were obtained from Tulane University Hospital between 2007–2009. 39 cases represented TNBC (33-African Americans, 4-Caucasians, 2-unknowns) and 40 cases represented ER+BC (21-African Americans, 16-Caucasians, 3-unknowns). Immunohistochemistry was used to measure staining distribution and intensity of total Src and activated phospho-SrcY416 (p-Y416Src) in carcinoma tissue and adjacent normal mammary ducts. In TNBC and ER+BC, total Src was significantly higher in cancer compared to adjacent normal ducts (P<0.0001) in both cell membrane and cytoplasm. In membranes, p-Y416Src was elevated in cancer compared to normal ducts. Total Src in the tumor cytoplasm was significantly higher in TNBC compared to ER+BC (P = 0.0028); conversely, p-Y416Src in the tumor cell membranes was higher in TNBC compared to ER+BC (P = 0.0106). Comparison between African American (n = 21) and Caucasian ER+BC (n = 16) revealed no significant difference in expression and localization of total Src and p-Y416Src. TNBC cases positive for lymph node metastasis showed elevated membrane p-Y416Src compared to lymph node negative TNBC (P = 0.027).

Conclusion/Significance

Total Src and p-Y416Src were expressed higher in cancer compared to adjacent normal ducts. Cytoplasmic total Src and membrane p-Y416Src were significantly higher in TNBC compared to ER+BC. TNBC cases with lymph node metastasis showed elevated membrane p-Y416Src. Taken together, Src was elevated in the membrane and cytoplasm of more aggressive TNBC.     

Claudin 1 inhibits cell migration and increases intercellular adhesion in triple-negative breast cancer cell line

Triple-negative “claudin 1 low” subtype represents around 15% of breast cancer and displays poor prognosis. The loss of claudin 1 is correlated with increased invasiveness and higher recurrence of the disease. Claudin 1 constitutes the backbone of the tight junction and is involved in cell-cell adhesion and migration processes. However, studies showed a controversial role of claudin 1 in cell migration. In this study, we aimed to clarify the effect of claudin 1 on migration of mesenchymal triple-negative breast cancer cells (TNBC). We reported that transient over expression of claudin 1 in MDA-MB-231 and Hs578T “claudin 1 low” TNBC cells inhibited cell migration using wound healing and transwell migration assays. In order to investigate more specifically the involvement of claudin 1, we generated stable MDA-MB-231 clones overexpressing claudin 1. Interestingly, the level of claudin 1 was correlated to the inhibition of cell migration and to the increase of cell-cell aggregation associated with enhanced formation of β-catenin adherens junction and occludin tight junction. Finally, we reported for the first time the key role of claudin 1 in the inhibition of cell migration process associated with the disappearance of stress fibers. These data suggest that re-expression of claudin 1 could be a promising strategy for regulating the migration of TNBC which no longer express claudin 1.

     

Antrodia camphorata inhibits epithelial-to-mesenchymal transition by targeting multiple pathways in triple-negative breast cancers

Antrodia camphorata (AC) exhibits potential for engendering cell-cycle arrest as well as prompting apoptosis and metastasis inhibition in triple-negative breast cancer (TNBC) cells. We performed the current study to explore the anti-epithelial-to-mesenchymal transition (EMT) properties of fermented AC broth in TNBC cells. Our results illustrated that noncytotoxic concentrations of AC (20–60 μg/ml) reversed the morphological changes (fibroblastic-to-epithelial phenotype) as well as the EMT by upregulating the observed E-cadherin expression. Furthermore, we discovered treatment with AC substantially inhibit the Twist expression in human TNBC (MDA-MB-231) cells as well as in those that were transfected with Twist. In addition, we determined AC to decrease the observed Wnt/β-catenin nuclear translocation through a pathway determined to be dependent on GSK3β. Notably, AC treatment consistently inhibited the EMT by downregulating mesenchymal marker proteins like N-cadherin, vimentin, Snail, ZEB-1, and fibronectin; at that same time upregulating epithelial marker proteins like occludin and ZO-1. Bioluminescence imaging that was executed in vivo demonstrated AC substantially suppressed breast cancer metastasis to the lungs. Notably, we found that western blot analysis confirmed that AC decreased lung metastasis as demonstrated by upregulation of E-cadherin expression in biopsied lung tissue. Together with our results support the anti-EMT activity of AC, indicating AC as having the potential for acting as an anticancer agent for the treatment of human TNBC treatment.