Malignant Phenotype Of Breast Cancer Research Articles

Hyperglycemia induces PFKFB3 overexpression and promotes malignant phenotype of breast cancer through RAS/MAPK activation

BackgroundBreast cancer is the most common tumor in women worldwide. Diabetes mellitus is a global chronic metabolic disease with increasing incidence. Diabetes mellitus has been reported to positively regulate the development of many tumors. However, the specific mechanism of hyperglycemic environment regulating breast cancer remains unclear. PFKFB3 (6-phosphofructose-2-kinase/fructose-2, 6-bisphosphatase 3) is a key regulatory factor of the glycolysis process in diabetes mellitus, as well as a promoter of breast cancer. So, we want to explore the potential link between PFKFB3 and the poor prognosis of breast cancer patients with hyperglycemia in this study.MethodsCell culture was utilized to construct different-glucose breast cancer cell lines. Immunohistochemistry was adopted to analyze the protein level of PFKFB3 in benign breast tissues, invasive ductal carcinoma with diabetes and invasive ductal carcinoma without diabetes. The Kaplan–Meier plotter database and GEO database (GSE61304) was adopted to analyze the survival of breast cancer patients with different PFKFB3 expression. Western blot was adopted to analyze the protein level of PFKFB3, epithelial–mesenchymal transition (EMT)-related protein and extracellular regulated protein kinases (ERK) in breast cancer cells. Gene Set Cancer Analysis (GSCA) was utilized to investigate the potential downstream signaling pathways of PFKFB3. TargetScan and OncomiR were utilized to explore the potential mechanism of PFKFB3 overexpression by hyperglycemia. Transfections (including siRNAs and miRNA transfection premiers) was utilized to restrain or mimic the expression of the corresponding RNA. Cell functional assays (including cell counting, MTT, colony formation, wound-healing, and cell migration assays) were utilized to explore the proliferation and migration of breast cancer cells.ResultsIn this study, we demonstrated that the expression of PFKFB3 in breast cancer complicated with hyperglycemia was higher than that in breast cancer with euglycemia through cell experiment in vitro and histological experiment. PFKFB3 overexpression decreased the survival period of breast cancer patients and was correlated with a number of clinicopathological parameters of breast cancer complicated with diabetes. PFKFB3 promoted the proliferation and migration of breast cancer in a hyperglycemic environment and might be regulated by miR-26. In addition, PFKFB3 stimulated epithelial-mesenchymal transition of breast cancer in a hyperglycemic environment. In terms of downstream mechanism exploration, we predicted and verified the cancer-promoting effect of PFKFB3 in breast cancer complicated with hyperglycemia through RAS/MAPK pathway.ConclusionsIn conclusion, PFKFB3 could be overexpressed by hyperglycemia and might be a potential therapeutic target for breast cancer complicated with diabetes.

MiR-598-5p inhibits breast cancer tumor growth and lung metastasis by targeting PPAPDC1A.

This study aimed to explore the effects of PPAPDC1A on the malignant phenotype of breast cancer (BC) in vivo and in vitro. PPAPDC1A expression was examined in BC tissues and cell lines by real-time polymerase chain reaction and Western blot. In this article, cell proliferation was evaluated by Cell Counting Kit-8 assay and colony formation assay, and cell migration and invasion were evaluated by wound healing assay and transwell assays. Furthermore, in vivo cell growth and pulmonary metastasis experiments were also performed using nude mice. The results showed that compared with normal tissues and cells, the PPAPDC1A expression in BC tissues and cell lines were both significantly increased. The PPAPDC1A targeting sequence significantly inhibited the PPAPDC1A expression and cell proliferation, migration, and invasion. The results of xenograft showed that knockdown of PPAPDC1A inhibited tumor growth and lung metastasis of BC. Then, the Dual-Luciferase Reporter Assay confirmed that miR-598-5p targeted the regulation of PPAPDC1A expression. In addition, the miR-598-5p expression in BC tissues was lower than that in the normal tissues. The rescue experiment showed that PPAPDC1A overexpression reversed the inhibitory effect of miR-598-5p mimic on cell proliferation, migration, and invasion. In conclusion, PPAPDC1A was highly expressed in BC tissues and cell lines, and miR-598-5p inhibited the malignant phenotype of BC by targeting PPAPDC1A.

ZNF384–ZEB1 feedback loop regulates breast cancer metastasis

BackgroundBreast cancer has become the most frequently diagnosed cancer worldwide. Increasing evidence indicated that zinc finger proteins (ZNFs), the largest family of transcription factors, contribute to cancer development and progression. Although ZNF384 is overexpressed in several types of human cancer, the role of ZNF384 in breast cancer remains unknown. Therefore, our research focused on ZNF384 regulation of the malignant phenotype of breast cancer and the underlying molecular mechanisms.MethodsCCK-8 and colony formation assays were used to evaluate cell proliferation. Transwell and scratch assays were used to evaluate the cell migration and invasion. Chromatin immunoprecipitation (ChIP)-qPCR and luciferase reporter assays were used to confirm the target relationship between ZNF384 and zinc finger E-box binding homeobox 1 (ZEB1). Xenografts were used to monitor the targets in vivo effects.ResultsWe noted that ZNF384 was significantly overexpressed in breast cancer and highlighted the oncogenic mechanism of ZNF384. ZNF384 transactivated ZEB1 expression and induced an epithelial and mesenchymal-like phenotype, resulting in breast cancer metastasis. Furthermore, ZNF384 may be a target of miR-485-5p, and ZEB1 can up-regulate ZNF384 expression by repressing miR-485-5p expression. Together, we unveiled a feedback loop of ZNF384–ZEB1 in breast cancer metastasis.ConclusionsThe findings suggest that ZNF384 can serve as a prognostic factor and a therapeutic target for breast cancer patients.

RAMP2-AS1 inhibits CXCL11 expression to suppress malignant phenotype of breast cancer by recruiting DNMT1 and DNMT3B

BackgroundBreast cancer is the most common malignancy in women populations. MethodsRAMP2-AS1 and CXCL11 expression in breast cancer tissues and cells were determined using RT-qPCR or Western blot. RIP analysis confirmed the interaction between DNMT1, DNMT3B and RAMP2-AS1. ChIP assay verified that RAMP2-AS1 recruited DNMT1 and DNMT3B to the promoter region of CXCL11. FISH detected the sub-localization of RAMP2-AS1 in breast cancer cells. Bisulfite sequencing PCR (BSP) tested the methylation level of CXCL11. The cell viability, proliferation, migration and apoptosis were assessed by CCK-8, colony formation, transwell and flow cytometry assays, respectively. IHC was performed to evaluate the expression of Ki67, CXCL11, MMP2 in tumor tissues. ResultsThe level of RAMP2-AS1 was decreased in breast cancer tissues and cells, whereas CXCL11 was highly expressed. Patients with decreased RAMP2-AS1 had a poor prognosis. RAMP2-AS1 inhibited breast cancer cell malignant phenotype. Besides, RAMP2-AS1 regulated the methylation of CXCL11 by recruiting DNMT1 and DNMT3B to the promoter region of CXCL11. RAMP2-AS1 overexpression suppressed the malignant phenotype through CXCL11 and inhibited tumor growth in vivo. ConclusionRAMP2-AS1 suppresses breast cancer malignant phenotype via DNMT1 and DNMT3B mediated inhibition of CXCL11.

Pregnancy-associated plasma protein-A (PAPPA) promotes breast cancer progression

ABSTRACT Breast cancer is the most common malignancy in females and poses a significant health threat to women. Pregnancy-associated plasma protein-A (PAPPA) is highly expressed in pregnancy-associated breast cancer (PABC) tissues. In this study, we investigated the functional role of PAPPA in regulating the malignant phenotype of breast cancer. We first examined the expression level of PAPPA in PABC tissue and breast cancer cell lines using quantitative real-time polymerase-chain reaction (qRT-PCR) and western blot. Next, the functional role of PAPPA in breast cancer cells was validated by overexpression and knockdown experiments. Cell counting kit-8 (CCK-8) proliferation assay, 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay, wound healing and transwell invasion assay were used to examine cell proliferation, migration, and invasion ability. We further identified the microRNA target regulating PAPPA and studied its functional role. Finally, we examined the impact of PAPPA on the tumorigenesis and metastasis of breast cancer in mice model. Our study revealed that PAPPA was upregulated in PABC tissues and breast cancer cells. Overexpression of PAPPA promoted cell proliferation, motility, invasion, and epithelial–mesenchymal transition (EMT). We further identified miR-497-5p as a negative regulator of PAPPA, which suppressed cell proliferation, migration, invasion, and EMT in breast cancer cells. We also validated the oncogenic role of PAPPA in the mouse xenograft model. Collectively, our study suggests that PAPPA is an oncogenic protein highly expressed in PABC tissues and promotes breast cancer progression, which could serve as a novel therapeutic target for breast cancer.

Long Intergenic Non-Coding RNA LINC00922 Aggravates the Malignant Phenotype of Breast Cancer by Regulating the microRNA-424-5p/BDNF Axis [Retraction

[This retracts the article DOI: 10.2147/CMAR.S267665.].

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

SummaryProlactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Long Intergenic Non-Coding RNA LINC00922 Aggravates the Malignant Phenotype of Breast Cancer by Regulating the microRNA-424-5p/BDNF Axis.

PurposeLong intergenic non-coding RNA 922 (LINC00922) plays a critical role in the progression of lung cancer. In this study, we aimed to quantify LINC00922 expression in breast cancer and determine its influence on the malignant behavior of breast cancer cells in vitro and in vivo. We also investigated the mechanism by which LINC00922 affects the progression of breast cancer.Materials and MethodsReverse transcription-quantitative polymerase chain reaction was performed to quantify LINC00922 expression in breast cancer tissues and cell lines. The cell counting kit-8 assay, flow cytometry, Transwell migration and invasion assays, and tumor model assays were performed to determine the effects of LINC00922 deficiency on breast cancer cell proliferation, apoptosis, migration and invasion in vitro, and tumor growth in vivo, respectively. Furthermore, bioinformatics analysis was performed to predict the potential target microRNA of LINC00922. The prediction was further evaluated using luciferase reporter and RNA immunoprecipitation assays.ResultsLINC00922 was clearly overexpressed in breast cancer tissues and cell lines. LINC00922 depletion restricted breast cancer cell proliferation, migration, and invasion but induced cell apoptosis in vitro. Additionally, its knockdown evidently repressed tumor growth of breast cancer cells in vivo. Mechanistically, LINC00922 was demonstrated to serve as a molecular sponge for miR-424-5p in breast cancer cells. Furthermore, brain-derived neurotrophic factor (BDNF) was verified as a direct target of miR-424-5p in breast cancer cells, and BDNF expression was found to be positively regulated by LINC00922 through sponging miR-425-5p. Rescue experiments further revealed that the influences on breast cancer cell proliferation, apoptosis, migration, and invasion induced by LINC00922 silencing were abrogated by increasing the output of the miR-424-5p/BDNF axis.ConclusionThe LINC00922/miR-424-5p/BDNF pathway is implicated in the acceleration of the malignant behavior of breast cancer cells. These findings suggest that this pathway is a promising novel molecular target in breast cancer therapy.

CircCDYL Acts as a Tumor Suppressor in Triple Negative Breast Cancer by Sponging miR-190a-3p and Upregulating TP53INP1

BackgroundBreast cancer is the most commonly diagnosed cancer and the leading cause of cancer death among females. Circular RNAs (circRNAs) have been implicated in the initiation and development of cancer. Here, we explored the biological role and regulatory mechanism of circCDYL in breast cancer. Materials and MethodsThe expression and correlation of circCDYL/miR-190a-3p/TP53INP1 axis in breast cancer tissues and cells were determined by quantitative polymerase chain reaction and Western blot. Cell-counting Kit-8, colony formation, cell migration, and invasion assays were applied to investigate the biological roles of circCDYL in breast cancer development and progression. ResultsCircCDYL were down-regulated in breast cancer tissues and cells, the expression of which positively correlated with patients’ survival rate. CircCDYL worked as a “sponge,” binding to miR-190a-3p directly, which inhibited the expression of miR-190a-3p and relieved the inhibition of tumor suppressor gene TP53INP1. ConclusionCircCDYL promotes apoptosis and inhibits proliferation of the malignant phenotype of breast cancer through regulating miR-190a-3p/TP53INP1 axis, which suggests that circCDYL is a potential therapeutic target for breast cancer.

Bisecting N-Acetylglucosamine on EGFR Inhibits Malignant Phenotype of Breast Cancer via Down-Regulation of EGFR/Erk Signaling.

Glycosylation, the most prevalent and diverse post-translational modification of protein, plays crucial biological roles in many physiological and pathological events. Alteration of N-glycan has been detected during breast cancer progression. Among the specific N-glycan structures, bisecting N-Acetylglucosamine (GlcNAc) is a β1,4-linked GlcNAc attached to the core β-mannose residue, and is catalyzed by glycosyltransferase MGAT3. Bisecting GlcNAc levels were commonly dysregulated in different types of cancer. In this study, we utilized mass spectrometry and lectin microarray analysis to investigate aberrant N-glycans in breast cancer cells. Our data showed the decreased levels of bisecting GlcNAc and down-regulated expression of MGAT3 in breast cancer cells than normal epithelial cells. Using PHA-E (a plant lectin recognizing and combining bisecting GlcNAc) based enrichment coupled with nanoLC-MS/MS, we analyzed the glycoproteins bearing bisecting GlcNAc in various breast cancer cells. Among the differentially expressed glycoproteins, levels of bisecting GlcNAc on EGFR were significantly decreased in breast cancer cells, confirmed by immunostaining and immunoprecipitation. We overexpressed MGAT3 in breast cancer MDA-MB-231 cells, and overexpression of MGAT3 significantly enhanced the bisecting N-GlcNAc on EGFR and suppressed the EGFR/Erk signaling, which further resulted in the reduction of migratory ability, cell proliferation, and clonal formation. Taken together, we conclude that bisecting N-GlcNAc on EGFR inhibits malignant phenotype of breast cancer via down-regulation of EGFR/Erk signaling.

Rhein Derivative 4F Inhibits the Malignant Phenotype of Breast Cancer by Downregulating Rac1 Protein.

BackgroundTriple-negative breast cancer is a common malignant tumor with unfavorable prognosis affecting women worldwide; thus, there is an urgent need for novel therapeutic drugs with improved anti-tumor activity. Rac family small GTPase 1 (Rac1) plays an important role in malignant behavior and is a promising therapeutic target. We reported an anthraquinone compound, Rhein, and its derivative, 4F, and investigated their downregulation effects on Rac1 in breast cancer cells in vitro.MethodsThe inhibition of cell proliferation by derivative 4F was investigated in two breast cancer (MDA-MB-231 and MCF-7) and normal breast (MCF-10A) cell lines by cell counting kit-8 assay and growth curves. The role of 4F in cell migration and invasion and cytoskeletal change were assessed by Transwell chamber assay and F-actin staining, respectively. The affinity of Rhein and its derivative for Rac1 protein and the regulation of Rac1 promoter activity were evaluated by molecular docking software and luciferase reporter gene assay, respectively. Rac1 protein expression was determined by western blot assay.ResultsCompared to Rhein, derivative 4F more strongly inhibited breast cancer cell proliferation, migration, and invasion and also cause cytoskeletal changes like those in paclitaxel. Derivative 4F not only bound more stably to Rac1 but also inhibited Rac1 promoter activity in cells and downregulated Rac1 protein expression.ConclusionsRhein derivative 4F is a new anthraquinone compound with better anti-tumor activity than that of the lead compound Rhein in breast cancer. It down-regulated Rac1 expression and may be a small molecule inhibitor of Rac1.

Spheroid-Induced Epithelial-Mesenchymal Transition Provokes Global Alterations of Breast Cancer Lipidome: A Multi-Layered Omics Analysis.

Metabolic rewiring has been recognized as an important feature to the progression of cancer. However, the essential components and functions of lipid metabolic networks in breast cancer progression are not fully understood. In this study, we investigated the roles of altered lipid metabolism in the malignant phenotype of breast cancer. Using a spheroid-induced epithelial-mesenchymal transition (EMT) model, we conducted multi-layered lipidomic and transcriptomic analysis to comprehensively describe the rewiring of the breast cancer lipidome during the malignant transformation. A tremendous homeostatic disturbance of various complex lipid species including ceramide, sphingomyelin, ether-linked phosphatidylcholines, and ether-linked phosphatidylethanolamine was found in the mesenchymal state of cancer cells. Noticeably, polyunsaturated fatty acids composition in spheroid cells was significantly decreased, accordingly with the gene expression patterns observed in the transcriptomic analysis of associated regulators. For instance, the up-regulation of SCD, ACOX3, and FADS1 and the down-regulation of PTPLB, PECR, and ELOVL2 were found among other lipid metabolic regulators. Significantly, the ratio of C22:6n3 (docosahexaenoic acid, DHA) to C22:5n3 was dramatically reduced in spheroid cells analogously to the down-regulation of ELOVL2. Following mechanistic study confirmed the up-regulation of SCD and down-regulation of PTPLB, PECR, ELOVL2, and ELOVL3 in the spheroid cells. Furthermore, the depletion of ELOVL2 induced metastatic characteristics in breast cancer cells via the SREBPs axis. A subsequent large-scale analysis using 51 breast cancer cell lines demonstrated the reduced expression of ELOVL2 in basal-like phenotypes. Breast cancer patients with low ELOVL2 expression exhibited poor prognoses (HR = 0.76, CI = 0.67–0.86). Collectively, ELOVL2 expression is associated with the malignant phenotypes and appear to be a novel prognostic biomarker in breast cancer. In conclusion, the present study demonstrates that there is a global alteration of the lipid composition during EMT and suggests the down-regulation of ELOVL2 induces lipid metabolism reprogramming in breast cancer and contributes to their malignant phenotypes.

ZNF326 promotes a malignant phenotype of breast cancer by interacting with DBC1

The biological role and underlying mechanism of action of zinc-finger protein 326 (ZNF326) in malignant tumors, including breast cancer, are still not clear. In this study, we detected high expression of ZNF326 in breast cancer specimens (60/111, 54.1%) and breast cancer cell lines (7/7); the expression level of ZNF326 was inversely associated with advanced pTNM stage (P = 0.002), positive lymph node metastasis (P = 0.004), poor prognosis in patients with breast cancer (P = 0.0097), and ER/PR/Her2 status (P = 0.013). Meanwhile, the ectopic expression of ZNF326 significantly upregulated MMP7, EMT-related proteins (Snail and Slug), and cell cycle-related proteins (cyclinA2 and cyclinB1); downregulated E-cadherin expression; and promoted the proliferation and invasiveness of breast cancer cells both in vivo and in vitro. Mechanistically, co-immunoprecipitation and immunofluorescence assays both demonstrated that ZNF326 interacted with deleted in breast cancer-1 (DBC1) in breast cancer cells. Additionally, DBC1 knockdown eliminated the up-regulation of MMP7, EMT-related proteins, and cell cycle-related proteins as well as the enhanced proliferation and invasiveness induced by ZNF326. Therefore, we concluded that ZNF326 is highly expressed in breast cancer, is associated with poor prognosis, and plays a vital role in promoting the malignant phenotype of breast cancer cells by interacting with DBC1.

CircZNF609 promotes breast cancer cell growth, migration, and invasion by elevating p70S6K1 via sponging miR-145-5p.

BackgroundAccumulating evidence suggests that circular RNAs (circRNAs) play critical roles in carcinomas. However, the contributions of circRNAs to breast cancer remain unclear. Herein, we determined the role of circZNF609 in breast cancer.MethodsA total of 143 breast cancer and 38 normal tissues were collected to assess the expression of circZNF609 and its relationship with breast cancer prognosis. A series of in vitro and in vivo functional experiments were carried out to elucidate the role of circZNF609 in breast cancer progression and its underlying molecular mechanisms.ResultsCircZNF609 was markedly over-expressed in breast cancer tissues and cell lines, and high circZNF609 expression was closely associated with poor outcome. Silencing of circZNF609 inhibited the malignant phenotype of breast cancer in vitro and in vivo. Mechanistically, circ-ZNF609 served as a sponge of miR-145-5p to elevate p70S6K1 expression. Moreover, miR-145-5p overexpression or p70S6K1 knockdown abrogated the oncogenic effects of circZNF609 in breast cancer. In addition, clinically, a strong negative correlation was observed between the expression of circZNF609 and miR-145-5p in breast cancer tissues (r=–0.597, P<0.001), whereas a positive correlation between circZNF609 and p70S6K1 expression (r=0.319, P<0.001).ConclusionThese data suggest that circZNF609 contributes to breast cancer progression, at least partly, by modulating the miR-145-5p/p70S6K1 axis, and it may be a potential therapeutic target for breast cancer.

Human snoRNA-93 is processed into a microRNA-like RNA that promotes breast cancer cell invasion

Genetic searches for tumor suppressors have recently linked small nucleolar RNA misregulations with tumorigenesis. In addition to their classically defined functions, several small nucleolar RNAs are now known to be processed into short microRNA-like fragments called small nucleolar RNA-derived RNAs. To determine if any small nucleolar RNA-derived RNAs contribute to breast malignancy, we recently performed a RNA-seq-based comparison of the small nucleolar RNA-derived RNAs of two breast cancer cell lines (MCF-7 and MDA-MB-231) and identified small nucleolar RNA-derived RNAs derived from 13 small nucleolar RNAs overexpressed in MDA-MB-231s. Importantly, we find that inhibiting the most differentially expressed of these small nucleolar RNA-derived RNAs (sdRNA-93) in MDA-MB-231 cells results primarily in a loss of invasiveness, whereas increased sdRNA-93 expression in either cell line conversely results in strikingly enhanced invasion. Excitingly, we recently determined sdRNA-93 expressions in small RNA-seq data corresponding to 116 patient tumors and normal breast controls, and while we find little sdRNA-93 expression in any of the controls and only sporadic expression in most subtypes, we find robust expression of sdRNA-93 in 92.8% of Luminal B Her2+tumors. Of note, our analyses also indicate that at least one of sdRNA-93’s endogenous roles is to regulate the expression of Pipox, a sarcosine metabolism-related protein whose expression significantly correlates with distinct molecular subtypes of breast cancer. We find sdRNA-93 can regulate the Pipox 3′UTR via standard reporter assays and that manipulating endogenous sdRNA-93 levels inversely correlates with altered Pipox expression. In summary, our results strongly indicate that sdRNA-93 expression actively contributes to the malignant phenotype of breast cancer through participating in microRNA-like regulation.

G-protein-coupled receptor 81 promotes a malignant phenotype in breast cancer through angiogenic factor secretion

G-protein-coupled receptor 81 (GPR81) functions as a receptor for lactate and plays an important role in the regulation of anti-lipolytic effects in adipocytes. However, to data, a role for GPR81 in the tumor microenvironment has not been clearly defined. Here, GPR81 expression in breast cancer patients and several breast cancer cell lines was significantly increased compared with normal mammary tissues and cells. GPR81 knockdown resulted in impaired breast cancer growth and led to apoptosis both in vitro and in vivo. Furthermore, the inhibition of GPR81 signaling suppressed angiogenesis through a phosphoinositide 3-OH kinase (PI3K)/Akt-cAMP response element binding protein (CREB) pathway, which led to decreased production of the pro-angiogenic mediator amphiregulin (AREG). Overall, these findings identify GPR81 as a tumor-promoting receptor in breast cancer progression and suggest a novel mechanism that regulates GPR81-dependent activation of the PI3K/Akt signaling axis in tumor microenvironment.

Decreased Usage of Specific Scrib Exons Defines a More Malignant Phenotype of Breast Cancer With Worsened Survival

SCRIB is a polarity regulator known to be abnormally expressed in cancer at the protein level. Here we report that, in breast cancer, an additional and hidden dimension of deregulations exists: an unexpected SCRIB exon usage pattern appears to mark a more malignant tumor phenotype and significantly correlates with survival. Conserved exons encoding the leucine-rich repeats tend to be overexpressed while others are underused. Mechanistic studies revealed that the underused exons encode part of the protein necessary for interaction with Vimentin and Numa1, a protein which is required for proper positioning of the mitotic spindle. Thus, the inclusion/exclusion of specific SCRIB exons is a mechanistic hallmark of breast cancer, which could potentially be exploited to develop more efficient diagnostics and therapies.

CD24 suppresses malignant phenotype by downregulation of SHH transcription through STAT1 inhibition in breast cancer cells

Hedgehog (Hh) signaling has been found to be activated in breast cancer stem cells (BCSCs). However, the precise role of the BCSCs marker, CD24, remains unclear. Here, we describe a relationship between CD24 and Sonic Hedgehog (SHH), and reveal a role for this relationship in the induction of a malignant phenotype of breast cancer. CD24 siRNA-transfected breast cancer cells (BCCs) demonstrated higher expression of SHH and GLI1, increased anchorage-independent proliferation, and enhanced invasiveness and superior tumorigenicity compared with control. Conversely, CD24 forced-expressing BCCs possessed decreased SHH and GLI1 expression, anchorage-independent proliferation, and invasiveness. Suppression of SHH decreased invasiveness through inhibition of matrix metalloproteinase (MMP)-2 expression, GLI1 expression, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo in CD24 siRNA transfected BCCs. DNA microarray analysis identified STAT1 as a relationship between CD24 and SHH. CD24 siRNA-transfected BCCs with concurrent STAT1 inhibition exhibited decreased SHH expression, invasiveness, anchorage-independent proliferation, tumorigenicity, and tumor volume in vivo. These results suggest that CD24 suppresses development of a malignant phenotype by down-regulating SHH transcription through STAT1 inhibition. CD24 gene transfer or STAT1 inhibition may represent new effective therapeutic strategies to target refractory breast cancer.

Subtype-specific accumulation of intracellular zinc pools is associated with the malignant phenotype in breast cancer.

BackgroundZinc (Zn) hyper-accumulates in breast tumors and malignant cell lines compared to normal mammary epithelium. The mechanisms responsible for Zn accumulation and the consequence of Zn dysregulation are poorly understood.MethodsMicroarrays were performed to assess differences in the expression of Zn transporters and metallothioneins (MTs) in human breast tumors and breast cancer cell lines. Real-time PCR and immunoblotting were employed to profile Zn transporter expression in representative luminal (T47D), basal (MDA-MB-231), and non-malignant (MCF10A) cell lines. Zn distribution in human tumors was assessed by X-ray fluorescence imaging. Zn distribution and content in cell lines was measured using FluoZin-3 imaging, and quantification and atomic absorption spectroscopy. Functional consequences of ZnT2 over-expression in MDA-MB-231 cells including invasion, proliferation, and cell cycle were measured using Boyden chambers, MTT assays, and flow cytometry, respectively.ResultsGene expression profiling of human breast tumors and breast cancer cell lines identified subtype-specific dysregulation in the Zn transporting network. X-ray fluorescence imaging of breast tumor tissues revealed Zn hyper-accumulation at the margins of Luminal breast tumors while Zn was more evenly distributed within Basal tumors. While both T47D and MDA-MB-231 cells hyper-accumulated Zn relative to MCF10A cells, T47D cells accumulated 2.5-fold more Zn compared to MDA-MB-231 cells. FluoZin-3 imaging indicated that Zn was sequestered into numerous large vesicles in T47D cells, but was retained in the cytoplasm and found in fewer and larger, amorphous sub-cellular compartments in MDA-MB-231 cells. The differences in Zn localization mirrored the relative abundance of the Zn transporter ZnT2; T47D cells over-expressed ZnT2, whereas MDA-MB-231 cells did not express ZnT2 protein due to proteasomal degradation. To determine the functional relevance of the lack of ZnT2 in MDA-MB-231cells, cells were transfected to express ZnT2. ZnT2 over-expression led to Zn vesicularization, shifts in cell cycle, enhanced apoptosis, and reduced proliferation and invasion.ConclusionsThis comprehensive analysis of the Zn transporting network in malignant breast tumors and cell lines illustrates that distinct subtype-specific dysregulation of Zn management may underlie phenotypic characteristics of breast cancers such as grade, invasiveness, metastatic potential, and response to therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0486-y) contains supplementary material, which is available to authorized users.

Abstract S3-02: NSABP B-36: A randomized phase III trial comparing six cycles of 5-fluorouracil (5-FU), epirubicin, and cyclophosphamide (FEC) to four cycles of adriamycin and cyclophosphamide (AC) in patients (pts) with node-negative breast cancer

Abstract Background NSABP B-36 was originally designed as a 2X2 factorial randomized study to compare 6 cycles of FEC-100 with 4 cycles of standard AC with or without celecoxib in pts with node-negative breast cancer. The rationale for the trial was based on observations from other adjuvant trials suggesting that longer duration of anthracycline-based therapy may result in improved outcomes and also on accumulating evidence that prostaglandins may contribute to the malignant phenotype in breast cancer. The trial opened in May 2004 but random assignment to celecoxib v placebo was terminated in December 2004 (after 327 pts were enrolled) because of concerns for increased risk of cardiovascular disease with the use of COX-2 inhibitors. The trial continued as a two-arm study and completed accrual in July 2008. The primary endpoint of disease-free survival (DFS), and secondary endpoints of overall survival (OS), recurrence-free interval (RFI), distant recurrence-free interval (DRFI), and adverse events comparing FEC-100 and AC are reported here. Analyses of quality of life indicators will be reported separately. Methods 2,722 pts with T1-3, pN0 breast cancer were randomly assigned to either adriamycin 60 mg/m2 and cyclophosphamide 600mg/m2 every 21 days for 4 cycles (n=1361) or 5-FU 500mg/m2, epirubicin 100 mg/m2 and cyclophosphamide 500 mg/m2 every 21 days for 6 cycles (n=1361). Hormone- receptor positive pts were to receive physician's choice of hormonal therapy for a minimum of 5 yrs. Trastuzumab for HER2-positive pts was at investigator's discretion but was not to begin for &amp;gt;3 weeks after the last dose of chemotherapy. All women treated with lumpectomy were to receive breast radiotherapy; post-mastectomy chest wall radiotherapy was at physician's discretion. The differences in DFS (OS, RFI, and DRFI) between two treatment arms were assessed by log-rank test stratified by hormone receptor status and type of surgery. Results Median follow-up is 82.8 months. Pt and tumor characteristics were equally distributed between the two groups (&amp;lt;50 years old: 40%, lumpectomy: 68%, and hormone positivity: 65 %). Overall, Grade 3 and 4 expected toxicities were more frequent in the FEC arm. Combined Grade 3/4 toxicities reaching statistical significance with a difference of 3% or more between AC and FEC arms included fatigue 3.55% v 8.45%, febrile neutropenia 3.70% v 9.42%, and thrombocytopenia 0.74% v 4.41%, respectively. While on treatment, Grade 3 left ventricular systolic dysfunction occurred in one pt in each arm. There were 2 toxicity deaths on AC and 5 on the FEC arm. Primary and secondary endpoint analyses at 8 ys did not reveal any significant differences in DFS, OS, RFI, or DRFI. EndpointAC (%)FEC (%)HRCIp-valueDFS83.082.81.040.85,1.260.70OS91.292.00.940.71,1.240.65RFI90.090.40.970.76,1.260.84DRFI92.393.00.890.66,1.200.43 Conclusions Six cycles of the FEC-100 regimen did not result in any efficacy advantage over 4 cycles of standard AC in node-negative breast cancer pts. As anticipated, the FEC-100 regimen resulted in greater toxicity. Support NCI: U10-CA-12027, -37377, -69974, -69651, -44066-26; Pharmacia &amp; Upjohn Co. Citation Format: Jacobs A Samuel, John W Wilson, Hanna Bandos, Richard M Elledge, André Robidoux, Louis Fehrenbacher, Patrick J Ward, Johnathan Polikoff, Adam M Brufsky, Louise Provencher, Alexander HG Paterson, John T Hamm, Robert L Carolla, Luis Baez-Diaz, Priya Rastogi, Thomas B Julian, D Lawrence Wickerham, Sandra M Swain, Charles E Geyer Jr, Eleftherios P Mamounas, Norman Wolmark. NSABP B-36: A randomized phase III trial comparing six cycles of 5-fluorouracil (5-FU), epirubicin, and cyclophosphamide (FEC) to four cycles of adriamycin and cyclophosphamide (AC) in patients (pts) with node-negative breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr S3-02.