Overexpressing Breast Cancer Cells Research Articles

The requirement for cyclin E in c-Myc overexpressing breast cancers

ABSTRACT Basal-like triple-negative breast cancers frequently express high levels of c-Myc. This oncoprotein signals to the core cell cycle machinery by impinging on cyclin E. High levels of E-type cyclins (E1 and E2) are often seen in human triple-negative breast tumors. In the current study, we examined the requirement for E-type cyclins in the c-Myc-driven mouse model of breast cancer (MMTV-c-Myc mice). To do so, we crossed cyclin E1- (E1-/-) and E2- (E2-/-) deficient mice with MMTV-c-Myc animals, and observed the resulting cyclin E1-/-/MMTV-c-Myc and cyclin E2-/-/MMTV-c-Myc females for breast cancer incidence. We found that mice lacking cyclins E1 or E2 developed breast cancers like their cyclin Ewild-type counterparts. In contrast, further reduction of the dosage of E-cyclins in cyclin E1-/-E2+/-/MMTV-c-Myc and cyclin E1+/-E2-/-/MMTV-c-Myc animals significantly decreased the incidence of mammary carcinomas, revealing arole for E-cyclins in tumor initiation. We also observed that depletion of E-cyclins in human triple-negative breast cancer cell lines halted cell cycle progression, indicating that E-cyclins are essential for tumor cell proliferation. In contrast, we found that the catalytic partner of E-cyclins, the cyclin-dependent kinase 2 (CDK2), is dispensable for the proliferation of these cells. These results indicate that E-cyclins, but not CDK2, play essential and rate-limiting roles in driving the proliferation of c-Myc overexpressing breast cancer cells.

24-Dehydrocholesterol reductase promotes the growth of breast cancer stem-like cells through the Hedgehog pathway.

Cholesterol is a risk factor for breast cancer. However, it is still unclear whether the cholesterol biosynthesis pathway plays any significant role in breast carcinogenesis. 24‐Dehydrocholesterol reductase (DHCR24) is a key enzyme in the cholesterol synthesis pathway. Although DHCR24 is reported to have different functions in different cancers, it is not clear whether DHCR24 is involved in breast cancer. In this study, we found that DHCR24 expression was higher in breast cancer especially in luminal and HER2 positive breast cancer tissues compared with normal breast. Changes in DHCR24 expression altered cellular cholesterol content without affecting the adherent growth of breast cancer cells. However, DHCR24 knockdown reduced whereas DHCR24 overexpression enhanced breast cancer stem‐like cell populations such as mammosphere and aldehyde dehydrogenase positive cell numbers. In addition, DHCR24 overexpression increased the expression of the Hedgehog pathway‐regulated genes. Treating DHCR24 overexpressing breast cancer cell lines with the Hedgehog pathway inhibitor GANT61 blocked DHCR24‐induced mammosphere growth and increased mRNA levels of the Hedgehog regulated genes. Furthermore, expression of a constitutively activated mutant of Smoothened, a key hedgehog signal transducer, rescued the decreases in mammosphere growth and Hedgehog regulated gene expression induced by knockdown of DHCR24. These results indicate that DHCR24 promotes the growth of breast cancer stem‐like cells in part through enhancing the Hedgehog signaling pathway. Our data suggest that cholesterol contribute to breast carcinogenesis by enhancing Hedgehog signaling and cancer stem‐like cell populations. Enzymes including DHCR24 involved in cholesterol biosynthesis should be considered as potential treatment targets for breast cancer.

Cytotoxic and Antimetastatic Activity of Hesperetin and Doxorubicin Combination Toward Her2 Expressing Breast Cancer Cells

Objective:This study aimed to explore Hesperetin (Hst) potency as a co-chemotherapeutics agent combined with Doxorubicin (Dox), particularly cytotoxic and antimetastasis effects toward MCF-7/HER2 cells. Methods:The cytotoxic effects were measured under MTT assay. The flowcytometry analysis was used to examine the cell cycle modulation and apoptosis evidence, while the effect of migration was assayed by scratch wound healing assay. Western blotting and gelatin zymography were carried out to examine the expression level of proteins, HER2, and Rac1. Results:Under MTT assay, Hst and Dox exhibited to decrease cell viability in a dose-dependent manner with the IC50 value of 377 and 0,8 µM, respectively. The combination of Hst and Dox at the respective doses of 95 and 0,2 µM showed a synergistic effect with the combination index of 0,63. Flow cytometry analysis of Hst-Dox revealed that those compounds caused cell cycle arrest at the G2/M phase and induced apoptosis. Hst also decreased HER2 and Rac1 expression, as shown by western blot. Hst inhibited lamellipodia formation and cell migration, as indicated by microscopic observation and wound healing scratch assay. The antimetastatic activity of Hst was associated with the reduction of Rac1 and MMP9 expression as measured by gelatine zymography assay. Conclusion:These results indicated that the combination of Hst and Dox-induced cell cycle arrest, apoptosis, decreased HER2, Rac1, MMP9 expression, and cell migration. Thus, Hst may have the potential to be developed as a co-chemotherapeutic agent combined with doxorubicin toward HER2 overexpressing breast cancer cells.

Extracellular Acidosis Promotes Metastatic Potency via Decrease of the BMAL1 Circadian Clock Gene in Breast Cancer.

Circadian oscillation is an essential process that influences many physiological and biological mechanisms and a decrease of circadian genes is associated with many diseases such as cancer. Despite many efforts to identify the detailed mechanism for decreasing circadian genes and recovering reduced circadian genes in cancer, it is still largely unknown. We found that BMAL1 was reduced in tumor hypoxia-induced acidosis, and recovered by selectively targeting acidic pH in breast cancer cell lines. Surprisingly, BMAL1 was reduced by decrease of protein stability as well as inhibition of transcription under acidosis. In addition, melatonin significantly prevented acidosis-mediated decrease of BMAL1 by inhibiting lactate dehydrogenase-A during hypoxia. Remarkably, acidosis-mediated metastasis was significantly alleviated by BMAL1 overexpression in breast cancer cells. We therefore suggest that tumor hypoxia-induced acidosis promotes metastatic potency by decreasing BMAL1, and that tumor acidosis could be a target for preventing breast cancer metastasis by sustaining BMAL1.

ATF3 Demethylation Promotes the Transcription of ARL4C, Which Acts as a Tumor Suppressor in Human Breast Cancer.

IntroductionBreast cancer is a common malignancy in females worldwide. In this study, we investigated the role of activating transcription factor 3 (ATF3) and ADP-ribosylation factor like-4 (ARL4) in human breast cancer, and the associated mechanisms.Materials and MethodsWe measured ATF3 and ATL4C expressions in 15 paired breast cancer tissues using qRT-PCR, Western blotting and IHC. Cell growth, migration and invasion were tested in ATF3 or ARL4C overexpression breast cancer cells. TCGA database analysis was done to identify the correlation between ATF3 and ARL4C. We evaluated the binding of ATF3 to ARL4C promoter sequences and the effect of hypermethylation and demethylation of ATF3. A meta-analysis was done to investigate the relationship between the expression of ATF3 and/or ARL4C and the poor prognoses.ResultsOur results showed that ATF3 and ARL4C were decreased in breast cancer specimens at both mRNA and protein levels. Restoration of ATF3 or ARL4C reduced breast cancer tumorigenesis, evidenced by decreased cell growth, migration and invasion. The expression of ATF3 was positively correlated with ARL4C in breast cancer specimens, and ATF3 was shown to bind to the ARL4C promoter sequences. Furthermore, the expression of ATF3 was negatively regulated by hypermethylation, and demethylation of ATF3 stimulated ATF3 expression, which further promoted ARL4C transcription. Finally, a meta-analysis showed that patients with breast cancer with lower expression levels of ATF3 and/or ARL4C had worse prognoses.ConclusionOur results suggest that the ATF3/ARL4C axis may be a prospective biomarker for diagnosis and determination of prognosis, and a potential target for breast cancer treatment.

Dual in vitro invasion/migration suppressing and tamoxifen response modulating effects of a recombinant anti-ALCAM scFv on breast cancer cells.

It has been shown that overexpression of activated leukocyte cell adhesion molecule (ALCAM) is involved in development of resistance to tamoxifen therapy and promotion of cell invasion, migration and metastasis in ER+ breast cancer cells. Thus, we hypothesized that blockade of ALCAM interconnections with antibodies could be an effective approach for reversing mentioned negative events associated with ALCAM overexpression in breast cancer cells. Here, an anti-ALCAM scFv was recombinantly expressed and used throughout study for examination of the putative anticancer effects of ALCAM blockade. The anti-ALCAM scFv coding sequence was obtained from GenBank database and after addition of a 6× His-tag moiety, signal peptide and flanking sequences, the whole construct was expressed in Escherichia coli. Tamoxifen resistant MCF7 cells were then pretreat for 24 hours with purified recombinant anti-ALCAM scFv prior to administration of tamoxifen. In parallel, the cytotoxicity profile of anti-ALCAM scFv and tamoxifen co-treatments against tamoxifen resistant and sensitive MCF7 cell lines was also evaluated using CompuSyn software. The invasion/migration inhibitory effects of anti-ALCAM scFv on MDA-MB-231 cells were also evaluated. Pretreatment with anti-ALCAM scFv could successfully enhance anti-proliferative effects of tamoxifen against resistant MCF-7 cell lines. Furthermore, the combination of 19.2:1 of tamoxifen to anti-ALCAM scFv demonstrated synergistic cell inhibitory effect against tamoxifen resistant MCF7 cell lines. Also, incubating MDA-MB-231 cell lines with anti-ALCAM scFv resulted in a 30% and 25% reduction in number of invaded and migrated cells respectively. Overall, application of anti-ALCAM scFv could significantly suppress cancer cells metastasis in vitro and modulate tamoxifen resistant ER+ MCF7 cell line's sensitivity to tamoxifen. SIGNIFICANCE OF THE STUDY: Acquisition of resistance to tamoxifen therapy is one of the major challenges associated with cancer chemotherapy, gradually turning a responsive tumour into a refractory more invasive one which ultimately ends in disease progression and relapse. Here, we reported expression of an anti-ALCAM scFv, capable of increasing the sensitivity of tamoxifen resistant ER+ MCF-7 cells to tamoxifen therapy following a 24-hour pretreatment period. In addition, we demonstrated that the anti-ALCAM scFv monotherapy was also capable of suppressing invasion and migration of MDA-MB-231 cells in Boyden chamber assays.

Overexpression of HMGA2 in breast cancer promotes cell proliferation, migration, invasion and stemness

ABSTRACTDespite improved therapeutic strategies for early-stage breast cancer, the most common cancer type in women, relapse remains common and the underlying mechanisms for this progression remain poorly understood. To gain more insight, we studied the DNA-binding protein HMGA2 in breast cancer development and stemness. We demonstrated that HMGA2 is overexpressed in breast cancer tissues at the mRNA and protein levels (P value <0.0001). HMGA2 knockdown and overexpression in breast cancer cells revealed that HMGA2 promotes cell proliferation and protects against apoptosis via the intrinsic pathway. HMGA2 knockdown also causes cell cycle arrest in G2/M phase. In addition, we found that HMGA2 increases breast cancer cell migration and invasion (P value <0.001) and promotes the acquisition of cancer stem cell features, both in vitro, in colony formation (P value <0.01) and spheroid assays, and in breast cancer tissues. Overexpression of HMGA2 in breast cancer spurs the acquisition of several hallmarks of cancer, including increased cell proliferation, migration, invasion and stemness, and decreased apoptosis. Thus, targeting HMGA2 could represent an effective strategy to block breast cancer progression.

Abstract P6-16-08: Evaluation of cytochrome P450 4A11 overexpression in breast cancer cells in vitro and in vivo

Abstract Metastatic breast cancer (BC) is a highly invasive form of cancer that contributes greatly to patient mortality. Due to the complex mechanisms underlying cancer growth and metastasis, novel therapeutic targets must be found to increase patient survival. One such target is the cytochrome P450 4 (CYP4) metabolic pathway leading to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a lipid signaling molecule that promotes both tumor cell proliferation and neovascularization in tumors by inducing the proliferation and migration of endothelial cells, therefore promoting BC tumor survival. N-hydroxy-N’-(4-butyl-2 methyl phenyl) formamidine (HET0016) is a selective inhibitor of 20-HETE synthesis, thus impairing tumor growth and neovascularization. This study aims to investigate changes in BC (MDAMB231 and MCF7) cell lines overexpressing CYP4A11, a human CYP4 enzyme, with and without HET0016 treatment. To accomplish this, cells that overexpressed CYP4A11 were produced using lentivirus infection and confirmed via western blot. Tumor cell aggressiveness was assessed in vitro using migration and proliferation assays. Tumor growth with and without CYP4A11 overexpression was also measured in vivo using athymic nude mice. The results demonstrated that cells overexpressing CYP4A11 showed protein levels 4 times higher than the non-modified cells. MDAMB231 is a triple-negative BC (TNBC) that, when overexpressing CYP4A11, demonstrated increased proliferation capacity and significant increased tumor growth compared to non-modified cells. TNBC cells overexpressing CYP4A11 also showed increased motility. However, modified MCF7 cells did not show significant alterations when compared to non-modified MCF7 cells. Additionally, all cells treated with HET0016 showed decreased migration in the first 24 hours. In conclusion, these results demonstrate that the overexpression of CYP4A11 in TNBC cells increased tumor aggressiveness, proving this pathway to be an important target for therapeutic inhibition. Citation Format: Sehar Ali, Thaiz Borin, Ali Arbab. Evaluation of cytochrome P450 4A11 overexpression in breast cancer cells in vitro and in vivo [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-16-08.

Abstract PD7-01: Identification of a high FOXA1-induced pro-metastatic enhancer signature in endocrine-resistant and metastatic breast cancer

Abstract Background: Forkhead box A1 (FOXA1) is a pioneer transcription factor (TF) for chromatin binding and function of other lineage-specific TFs essential for the normal development of endoderm-derived organs. Aberrant FOXA1 signaling, due to genetic amplification or mutations and/or overexpression, has been frequently detected in metastatic tumors of the breast, prostate, pancreas, bladder and thyroid, suggesting a general role and mechanism of FOXA1-driven tumorigenesis and disease progression. We recently reported that high levels of FOXA1 (H-FOXA1) promote endocrine-resistant (EndoR) and metastatic phenotypes in estrogen receptor (ER)+ breast cancer (BC) cells. Here we sought to uncover the role and the mechanisms by which H-FOXA1 promotes EndoR metastatic BC. Methods: Genomic sequencing data from an ER+/HER2- metastatic BC cohort (n=781, MSK-IMPACT; cBioportal) were used to compare mutations and copy number alterations of FOXA1 and ESR1. Genome-wide FOXA1-chromatin binding (cistrome) and distribution of the enhancer marks histone H3 lysine 27 acetylation (H3K27ac) and lysine 4 mono-methylation (H3K4me1) were analyzed by ChIP-seq in MCF7 cell model with inducible H-FOXA1. FOXA1 cistrome, H3K27ac distribution, and transcriptome of a FOXA1-overexpressing pancreatic ductal adenocarcinoma cell model (PDA-hT2) were obtained from NCBI GEO (GSE99311). The core regulatory circuitry (CRC) Mapper was used to identify auto-regulatory loop of TFs induced by H-FOXA1. Gene Ontology was used for gene set functional annotation. FOXA1-associated enhancers of ER+ metastatic vs. primary tumors were analyzed using the H3K27ac epigenome data (European Nucleotide Archive, PRJEB22757). Results: The FOXA1 and ESR1 genetic amplification and mutations displayed a largely mutually exclusive pattern in ER+/HER2- metastatic BC, suggesting a role of hyperactive FOXA1 signaling in promoting EndoR and metastatic BC distinct from that of the ESR1 mutations. FOXA1 overexpression in BC cells resulted in increased FOXA1 DNA binding and the establishment of more regions with gained H3K27ac and/or H3K4me1, suggesting a more accessible and active chromatin state. H-FOXA1-induced upregulated genes were enriched for the gained H3K27ac or H3K4me1, especially for the enhancers with both marks. An enhancer signature with gained and overlapped H3K27ac and H3K4me1 predicts genes enriched for proliferation, anti-apoptosis and developmental signaling. Upregulated genes induced by H-FOXA1 with gained enhancers were further enriched for pro-metastatic processes, sharing the same characteristics of cellular morphogenesis during embryonic development. Similar results were obtained using integrated data from the PDA-hT2 cell model, sharing enriched pro-metastatic genes predicted by the H-FOXA1-induced enhancer signature. A CRC auto-regulatory TF loop, comprising components of the AP-1 and SMAD families, was predicted to amplify the impact of this enhancer signature on activation of the pro-metastatic transcriptional programs. In line with our preclinical findings, epigenetic changes of active enhancers in ER+ metastatic vs. primary BC were associated with the H-FOXA1-induced enhancer signature. Conclusions: Our study suggests that in ER+ metastatic BC, genetic alterations of FOXA1 leading to hyperactive FOXA1 signaling involves epigenetic evolution to promote a pro-metastatic enhancer signature. This genome-wide H-FOXA1-induced enhancer signature supports the role of H-FOXA1 in unleashing oncogenic activities of lineage-specific TFs in many types of metastatic tumors. Developing therapeutics targeting FOXA1 itself or key components of the H-FOXA1-induced CRC is warranted to treat or prevent EndoR and metastatic BC effectively via targeting the entire aberrant transcriptional programs. Citation Format: Xiaoyong Fu, Resel Pereira, Carmine De Angelis, Sarmistha Nanda, Lanfang Qin, Jamunarani Veeraraghavan, Pier Selenica, Britta Weigelt, Jorge S Reis-Filho, Agostina Nardone, Rinath Jeselsohn, Myles Brown, Mothaffar F Rimawi, C Kent Osborne, Rachel Schiff. Identification of a high FOXA1-induced pro-metastatic enhancer signature in endocrine-resistant and metastatic breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-01.

Abstract P6-05-08: Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers

Abstract Introduction: Increased expression of progesterone receptor membrane component 1 (PGRMC1), has been linked to breast cancer cell proliferation and tumor growth via epidermal growth factor receptor (EGFR) dependent interaction. As estrogen receptor positive breast cancers receive endocrine therapy and triple negative breast cancers (TNBC) rely on chemotherapeutic agents and or small molecule inhibitors. PGRMC1 could be a potential therapeutic target for both estrogen receptor positive and TNBCs, however its mechanism of action remains elusive. We, aim to understand the signaling mechanism behind PGRMC1 and its potential as viable target for the treatment of breast cancers. Materials and Methods: A panel of non-malignant and malignant breast cell lines were cultured and screened for PGRMC1 expression. PGRMC1 overexpressing breast cancer cell lines were treated with AG-205 (PGRMC1 inhibitor) and PGRMC1 targeting siRNAs. MTS, qRT-PCR, Western blot, immunofluorescence, flow cytometry assays and phospho explorer antibody array analysis were performed. Results: Increased PGRMC1 mRNA and protein levels were observed in estrogen receptor positive ZR-75-1 and TNBC MDA-MB-468 cells, these results were validated and compared to online RNA-seq based gene expression analysis of breast cell lines and breast tumor data sets. Data from online databases also demonstrated PGRMC1 overexpression in multiple breast cancer subtypes (Luminal, Basal B and Basal A). Immunohistochemistry demonstrated strong staining for PGRMC1 in human breast cancer tissue compared to normal tissue. Both AG-205 treatment and PGRMC1 silencing decreased cell proliferation, induced cell cycle arrest at the G0/G1 phase, promoted apoptosis and reduced the capability of the cells to migrate and invade. Phospho-specific antibody array data demonstrated overall downregulation of the EGFR/PI3K/AKT signaling mechanisms following AG-205 and PGRMC1 silencing. Alteration in the expression of key markers of cell proliferation, apoptosis and cell cycle revealed that PGRMC1 inhibition decreases breast cancer proliferation. Conclusion: Our data demonstrate that PGRMC1 plays a prominent role in regulating breast cancer growth and progression by altering the EGFR/PI3K/AKT signaling mechanisms. Citation Format: Diego A Pedroza, Alejandra Bencomo, Adriana Galvez, Ramadevi Subramani, Venkatesh Rajamanickam, Rajkumar Lakshmanaswamy. Progesterone receptor membrane component 1 controls cellular proliferation and plays a key role in the molecular circuitry of both ER positive and triple negative breast cancers [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-08.

Abstract P2-04-02: Progesterone receptor membrane component 1 - A novel key regulator in lipid homeostasis drives oncogenic signaling resulting in breast cancer progression

Abstract Background: PGRMC1 (progesterone receptor membrane component 1) is a highly conserved heme binding protein which is overexpressed especially in hormone receptor positive breast cancer and plays an important role in breast cancer carcinogenesis. Nevertheless, little is known about the mechanisms by which PGRMC1 drives tumor progression. Based on our previous studies on the involvement of PGRMC1 in cholesterol metabolism we describe a potential mechanism by which PGRMC1 can increase lipid metabolism and alter EGFR signaling. Methods: To obtain PGRMC1 overexpressing cell lines, MCF7, T47D and MDA cells were stably transfected with expression plasmid pcDNA3.1. containing HA-tagged PGRMC1. As a control, cells transfected with the empty plasmid were used.To investigate the role of PGRMC1 in breast cancer progression, expression of genes involved in lipid metabolism were quantified by qPCR. Subsequent lipid and cholesterol levels as well as lipid raft expression were measured by flow cytometry in various breast cancer cell lines overexpressing PGRMC1 compared to empty vector control. Since many studies show that EGFR is enriched in lipid rafts, we further investigated alteration of EGFR/HER2 signaling through PGRMC1 overexpression via western blot and immunofluorescence analyses. The impact of lipid inhibition on tumor progression was analyzed by viability assays. Results: PGRMC1 overexpression resulted in higher levels of mRNA coding for proteins responsible for lipid homeostasis (SREPF1, SREBF2), lipid uptake (LDLR) and lipid synthesis (FASN, HMGCS, SCD, ACAT) leading to higher levels of neutral lipids, estradiol and cholesterol. Furthermore, PGRMC1 overexpressing hormone receptor positive cells show increased lipid raft formation with higher expression of EGFR/HER2 and altered EGFR/HER2 signaling. PGRMC1 overexpressing cells are more sensitive to a simvastatin treatment, which indicates a higher dependence of PGRMC1 overexpressing cells on cholesterol synthesis. Conclusion: Cholesterol and lipid metabolism play an important role in breast carcinogenesis. Our recent studies underline the effects of PGRMC1 on increasing lipid synthesis and uptake thereby altering oncogenic signaling. PGRMC1 overexpressing breast cancer cells could be particularly suitable for a statin treatment. Citation Format: Hannah Asperger, Marina Ludescher, Nadia Stamm, Jan-Philipp Cieslik, Ute Hofmann, Ulrich Zanger, Zaklina Kovacevic, Des Richardson, Eugen Ruckhäberle, Tanja Fehm, Dieter Niederacher, Hans Neubauer. Progesterone receptor membrane component 1 - A novel key regulator in lipid homeostasis drives oncogenic signaling resulting in breast cancer progression [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-04-02.

Folate receptor alpha targeted delivery of artemether to breast cancer cells with folate-decorated human serum albumin nanoparticles

The pharmaceutical application of artemether (ARM) as an anticancer natural agent is hampered due to its poor solubility and bioavailability. In the present study, ARM was encapsulated in human serum albumin nanoparticles (HSA NPs) via desolvation method led to improvement of the water solubility by 50 folds. In further, folate-decorated ARM-HSA NPs (F-ARM-HSA NPs) were developed to enhance targeted delivery to folate receptor alpha (FRα)-overexpressing breast cancer cells. The hydrodynamic diameter and the zeta potential value of F-ARM-HSA NPs were 198 ± 11.22 nm and −23 ± 0.88 mV, respectively. Fluorescent microscopy demonstrated an enhanced cellular uptake of F-ARM-HSA NPs by high FRα-expressing MDA-MB-231 breast cancer cells compared to low FRα-expressing SK-BR-3 breast cancer cells. Cytotoxicity assay revealed a small significant difference between cytotoxicity effect of targeted and non-targeted NPs in SK-BR-3 cells. However, in MDA-MB-231 cells due to FRα-mediated endocytosis, the F-conjugated NPs had less inhibitory concentration (IC50) value (19.82 μg/mL) and higher cytotoxicity after 72 h compared to non-targeted ARM-HSA NPs. Flow cytometry analysis indicated a more potent drug-induced apoptosis rather than necrosis. The results suggest that our novel F-ARM-HSA NPs are likely to be recommended as a promising candidate for combination therapy of FRα-overexpressing breast cancer cells.

MiR-135-5p inhibits TGF-β-induced epithelial-mesenchymal transition and metastasis by targeting SMAD3 in breast cancer.

Breast cancer (BC) is the most frequently diagnosed malignant tumors and the leading cause of death due to cancer in women around the world. A growing body of studies have documented that microRNA (miR)-135-5p is associated with the development and progression of BC. Considering that sekelsky mothers against dpp3 (SMAD3) plays a crucial role in transforming growth factor (TGF)-β/SMAD pathway and epithelial-mesenchymal transition (EMT) process, it is critical to elucidate the crosstalk and underlying regulatory mechanisms between miR-135-5p and SMAD3 in controlling TGF-β-mediated EMT in BC metastasis. Our results revealed a reciprocal expression pattern between miR-135-5p and SMAD3 mRNA in BC tissues and cell lines. Moreover, miR-135-5p was decreased in BC tissues compared to adjacent breast tissues; more interesting, miR-135-5p mRNA levels (Tumor/Normal, T/N) was further decreased in BC patients with lymph node metastasis, while SMAD3 mRNA levels were increased. Gain- and loss-of-function assays indicated that overexpression of miR-135-5p inhibited TGF-β-mediated EMT and BC metastasis in vitro and in vivo. Furthermore, knockdown of SMAD3 produced a consistent phenotype of miR-135-5p overexpression in breast cancer cells. Mechanistically, SMAD3, a pivotal transcriptional modulator of TGF-β/SMAD pathway, for the first time, was analyzed and identified as a target gene of miR-135-5p by bioinformatic algorithms and dual-luciferase reporter assays. Taken together, we clarified that miR-135-5p suppressed TGF-β-mediated EMT and BC metastasis by negatively regulating SMAD3 and TGF-β/SMAD signaling. Our findings supported that miR-135-5p may serve as a tumor suppressor, and be a valuable diagnostic biomarker for the treatment of BC.

Low-density lipoprotein decorated and indocyanine green loaded silica nanoparticles for tumor-targeted photothermal therapy of breast cancer

Photothermal therapy (PTT) is well recognized to be a highly reliable way to treat cancer effectively. Nevertheless, the poor tumor targetability and the inferior stability of PTT agents are two remaining difficulties in PTT. As a result, in this study, the silica nanoparticles (SLN) were coated by low-density lipoprotein (LDL) to develop a multifunctional platform (LDL/SLN) to specifically target the LDL receptor (LDLR) overexpressed breast cancer cells. Furthermore, indocyanine green (ICG) was loaded inside LDL/SLN to establish a drug delivery system (DDS) fit for tumor-specific PTT of breast cancer. According to our results, LDL/SLN/ICG was nano-scaled particles with uniform size and reliable stability under physiological condition. Additionally, LDL/SLN/ICG could smartly target the MCF-7 cells that overexpress LDLR. Most importantly, further studies also demonstrated that LDL/SLN/ICG showed beneficial outcome than SLN/ICG. The high stability as well as the moderate size made it suitable to serve as DDS for cancer therapy. Moreover, the LDL modification was able to enhance the tumor accumulation of LDL/SLN/ICG, which finally achieved elevated PTT benefits than other groups. We therefore suggested that LDL/SLN/ICG could be a future alternative for promising tumor-targeted PTT for breast cancer.

LncRNA SNHG3 promotes cell proliferation and invasion through the miR-384/hepatoma-derived growth factor axis in breast cancer.

Long noncoding RNAs (lncRNAs) have been found to be abnormally expressed in cancer, and lncRNA small nucleolar RNA host genes (SNHGs) play critical roles in tumour progression. SNHG3 has been identified as an oncogene in multiple tumour types. However, the role of SNHG3 in breast cancer has not been reported. In this study, we found that SNHG3 was upregulated and associated with tumour malignancy in patients with breast cancer. SNHG3 knockdown inhibited the growth and metastatic capabilities of breast cancer cells in vitro and vivo. We used bioinformatics prediction and functional assay validation to determine that SNHG3 upregulation inhibited miR-384 activity and led to hepatoma-derived growth factor (HDGF) overexpression in breast cancer cells. The findings of this study show that SNHG3 functions as an oncogene in breast cancer and promotes breast cancer cell proliferation and invasion by regulating the miR-384/HDGF axis. The present study might provide a new target for the treatment of breast cancer.

MicroRNA‑133b targets TGFβ receptor I to inhibit TGF‑β‑induced epithelial‑to‑mesenchymal transition and metastasis by suppressing the TGF‑β/SMAD pathway in breast cancer.

Breast cancer (BC) is one of the most common types of cancer and the leading cause of cancer-associated mortality among women worldwide. Accumulating evidence indicates that microRNA (miR)-133b inhibits the proliferation and invasion of cancer cells. Considering that transforming growth factor (TGF)-β signaling plays a key role in cellular epithelial-to-mesenchymal transition (EMT) and cancer metastasis, it is crucial to explore the roles and underlying molecular mechanisms of miR-133b in regulating TGF-β-induced EMT during progression of BC. In the present study, an inverse correlation was observed between the expression of miR-133b and TGFβ receptor I (TGFβR1) mRNA in BC cells and tissues. Furthermore, miR-133b expression was found to be decreased in the BC tissues of patients with lymph node metastasis and advanced tumor-node-metastasis stage, while the expression of TGFβR1 was upregulated. Overexpression of miR-133b significantly decreased the expression of TGFβR1, an indispensable receptor of TGF-β/SMAD signaling, and suppressed TGF-β-induced EMT and BC cell invasion in vitro, whereas miR-133b knockdown exerted the opposite effects. Mechanistically, TGFβR1 was verified as a direct target of miR-133b as determined by bioinformatics analysis and a dual-luciferase reporter assay. In addition, small interfering RNA-mediated knockdown of TGFβR1 mimicked the phenotype of miR-133b overexpression in BC cells. Furthermore, miR-133b overexpression suppressed BC cell invasion in vivo. Collectively, the findings of the present study indicated that miR-133b acts as a tumor suppressor, inhibiting TGF-β-induced EMT and metastasis by directly targeting TGFβR1, and suppressing the TGF-β/SMAD pathway. Therefore, miR-133b may be of value as a diagnostic biomarker of BC.

Prolactin hormone exerts anti-tumorigenic effects in HER-2 overexpressing breast cancer cells through regulation of stemness.

Breast cancers characterized by HER2 overexpression, belong to HER-2 enriched or luminal B subtypes, are frequently associated with higher incidence of tumor recurrence and therapeutic failure. These aggressive features have been attributed to the presence of cancer stem-like cell subpopulations known to have high tumor initiation, self -renewal capacities and high metastatic potential. Depleting these stem-like cells in these tumors therefore might help in improving therapeutic response and patient outcome. Here we used human breast cancer cells representative of HER2- enriched and luminal B subtypes as well as purified ALDH-positive stem-like cell subpopulation for in vitro cell viability, proliferation, tumorshpere formation analyses and gene expression studies. In addition, we used a pre-clinical xenograft HER2 mouse model (NOD/SCID mice) for in vivo tumorigenesis assessment. Furthermore, patient survival outcomes were evaluated using in silico bioinformatics analyses of publicly available datasets. Our results indicate that prolactin (PRL) exerts anti-tumorigenic effects in HER-2 positive breast cancer cells. Importantly, PRL caused a significant reduction in ALDHhi stem-like subpopulation, as well as their viability and tumorsphere formation capacity. Molecularly we found PRL to suppress gene expression of markers involved in stemness, tumor initiation, drug resistance and poor patient outcome found to be enriched in the ALDHhi stem-like subpopulation. Furthermore, we show PRL to impede tumor growth of HER-2 xenografts and to suppress expression of Ki67 proliferative marker. Finally, we found PRL pathway gene signature to correlate with favorable patient outcomes in HER-2 and luminal B breast cancer patients. Together these results emphasize an anti-tumorigenic role with a potential therapeutic value for PRL in HER-2 and luminal B breast cancer subtypes targeting the cancer stem-like cells.

Cancer Testis Antigen Promotes Triple Negative Breast Cancer Metastasis and is Traceable in the Circulating Extracellular Vesicles

Triple negative breast cancer (TNBC) has poor survival, exhibits rapid metastases, lacks targeted therapies and reliable prognostic markers. Here, we examined metastasis promoting role of cancer testis antigen SPANXB1 in TNBC and its utility as a therapeutic target and prognostic biomarker. Expression pattern of SPANXB1 was determined using matched primary cancer, lymph node metastatic tissues and circulating small extracellular vesicles (sEVs). cDNA microarray analysis of TNBC cells stably integrated with a metastasis suppressor SH3GL2 identified SPANXB1 as a potential target gene. TNBC cells overexpressing SH3GL2 exhibited decreased levels of both SPANXB1 mRNA and protein. Silencing of SPANXB1 reduced migration, invasion and reactive oxygen species production of TNBC cells. SPANXB1 depletion augmented SH3GL2 expression and decreased RAC-1, FAK, A-Actinin and Vinculin expression. Phenotypic and molecular changes were reversed upon SPANXB1 re-expression. SPANXB1 overexpressing breast cancer cells with an enhanced SPANXB1:SH3GL2 ratio achieved pulmonary metastasis within 5 weeks, whereas controls cells failed to do so. Altered expression of SPANXB1 was detected in the sEVs of SPANXB1 transduced cells. Exclusive expression of SPANXB1 was traceable in circulating sEVs, which was associated with TNBC progression. SPANXB1 represents a novel and ideal therapeutic target for blocking TNBC metastases due to its unique expression pattern and may function as an EV based prognostic marker to improve TNBC survival. Uniquely restricted expression of SPANXB1 in TNBCs, makes it an ideal candidate for targeted therapeutics and prognostication.

Ibrutinib as a potential therapeutic option for HER2 overexpressing breast cancer - the role of STAT3 and p21.

Treatment response rates to current anticancer therapies for HER2 overexpressing breast cancer are limited andare associated withsevere adverse drug reactions. Tyrosine kinases perform crucial roles in cellular processes by mediating cell signalling cascades. Ibrutinib is a recently approved Tyrosine Kinase Inhibitor (TKI) that has been shown be an effective therapeutic option for HER2 overexpressing breast cancer. The molecular mechanisms, pathways, or genes that are modulated by ibrutinib and the mechanism of action of ibrutinib in HER2 overexpressing breast cancer remain obscure. In this study, we have performed a kinome array analysis of ibrutinib treatment in two HER2 overexpressing breast cancer cell lines. Our analysis shows that ibrutinib induces changes in nuclear morphology and causes apoptosis via caspase-dependent extrinsic apoptosis pathway with the activation of caspases-8, caspase-3, and cleavage of PARP1. We further show that phosphorylated STAT3Y705 is upregulated and phosphorylated p21T145 is downregulated upon ibrutinib treatment. We propose that STAT3 upregulation is a passive response as a result of induction of DNA damage and downregulation of phosphorylated p21 is promoting cell cycle arrest and apoptosis in the two HER2 overexpressing cell lines. These results suggest that inhibitors of STAT3 phosphorylation may be potential options for combination therapy to help increase the efficacy of ibrutinib against HER2-overexpressing tumors.

LncRNA NEAT1 Silenced miR-133b Promotes Migration and Invasion of Breast Cancer Cells.

Breast cancer, the most prevalent cancer type among women worldwide, remains incurable once metastatic. Long noncoding RNA (lncRNA) and microRNA (miRNA) play important roles in breast cancer by regulating specific genes or proteins. In this study, we found miR-133b was silenced in breast cancer cell lines and in breast cancer tissues, which predicted poor prognosis in breast cancer patients. We also confirmed that lncRNA NEAT1 was up-regulated in breast cancer and inhibited the expression of miR-133b, and identified the mitochondrial protein translocase of inner mitochondrial membrane 17 homolog A (TIMM17A) that serves as the target of miR-133b. Both miR-133b knockdown and TIMM17A overexpression in breast cancer cells promoted cell migration and invasion both in vitro and in vivo. In summary, our findings reveal that miR-133b plays a critical role in breast cancer cell metastasis by targeting TIMM17A. These findings may provide new insights into novel molecular therapeutic targets for breast cancer.