Proteins In Breast Cancer Cells Research Articles

Short Communication: Novel Di- and Triselenoesters as Effective Therapeutic Agents Inhibiting Multidrug Resistance Proteins in Breast Cancer Cells.

Breast cancer has the highest incidence rate among all malignancies worldwide. Its high mortality is mainly related to the occurrence of multidrug resistance, which significantly limits therapeutic options. In this regard, there is an urgent need to develop compounds that would overcome this phenomenon. There are few reports in the literature that selenium compounds can modulate the activity of P-glycoprotein (MDR1). Therefore, we performed in silico studies and evaluated the effects of the novel selenoesters EDAG-1 and EDAG-8 on BCRP, MDR1, and MRP1 resistance proteins in MCF-7 and MDA-MB-231 breast cancer cells. The cytometric analysis showed that the tested compounds (especially EDAG-8) are inhibitors of BCRP, MDR1, and MRP1 efflux pumps (more potent than the reference compounds-novobiocin, verapamil, and MK-571). An in silico study correlates with these results, suggesting that the compound with the lowest binding energy to these transporters (EDAG-8) has a more favorable spatial structure affecting its anticancer activity, making it a promising candidate in the development of a novel anticancer agent for future breast cancer therapy.

Bag-1-mediated HSF1 phosphorylation regulates expression of heat shock proteins in breast cancer cells.

According to the World Health Organization in 2022, 2.3 million women were diagnosed with breast cancer. Investigating the interaction networks between Bcl-2-associated athanogene (Bag)-1 and other chaperone proteins may further the current understanding of the regulation of protein homeostasis in breast cancer cells and contribute to the development of treatment options. The present study aimed to determine the interactions between Bag-1 and heat shock proteins (HSPs); namely, HSP90, HSP70 and HSP27, to elucidate their role in promoting heat shock factor-1 (HSF1)-dependent survival of breast cancer cells. HER2-negative (MCF-7) and HER2-positive (BT-474) cell lines were used to examine the impact of Bag-1 expression on HSF1 and HSPs. We demonstrated that Bag-1 overexpression promoted HER2 expression in breast cancer cells, thereby resulting in the concurrent constitutive activation of the HSF1-HSP axis. The activation of HSP results in the stabilization of several tumor-promoting HSP clients such as AKT, mTOR and HSF1 itself, which substantially accelerates tumor development. Our results suggest that Bag-1 can modulate the chaperone activity of HSPs, such as HSP27, by directly or indirectly regulating the phosphorylation of HSF1. This modulation of chaperone activity can influence the activation of genes involved in cellular homeostasis, thereby protecting cells against stress.

An 8-aminoquinoline-naphthyl copper complex causes apoptotic cell death by modulating the expression of apoptotic regulatory proteins in breast cancer cells

Breast cancer is one of the most common cancers globally and a leading cause of cancer-related deaths among women. Despite the combination of chemotherapy with targeted therapy, including monoclonal antibodies and kinase inhibitors, drug resistance and treatment failure remain a common occurrence. Copper, complexed to various organic ligands, has gained attention as potential chemotherapeutic agents due to its perceived decreased toxicity to normal cells. The cytotoxic efficacy and the mechanism of cell death of an 8-aminoquinoline-naphthyl copper complex (Cu8AqN) in MCF-7 and MDA-MB-231 breast cancer cell lines was investigated. The complex inhibited the growth of MCF-7 and MDA-MB-231 cells with IC50 values of 2.54 ± 0.69 μM and 3.31 ± 0.06 μM, respectively. Nuclear fragmentation, annexin V binding, and increased caspase-3/7 activity indicated apoptotic cell death. The loss of mitochondrial membrane potential, an increase in caspase-9 activity, the absence of active caspase-8 and a decrease of tumour necrosis factor receptor 1(TNFR1) expression supported activation of the intrinsic apoptotic pathway. Increased ROS formation and increased expression of haem oxygenase-1 (HMOX-1) indicated activation of cellular stress pathways. Expression of p21 protein in the nuclei was increased indicating cell cycle arrest, whilst the expression of inhibitor of apoptosis proteins (IAPs); cIAP1, XIAP and survivin were decreased, creating a pro-apoptotic environment. Phosphorylated p53 species; phospho-p53(S15), phospho-p53(S46), and phospho-p53(S392) accumulated in MCF-7 cells indicating the potential of Cu8AqN to restore p53 function in the cells. In combination, the data indicates that Cu8AqN is a useful lead molecule worthy of further exploration as a potential anti-cancer drug.

The immunotoxin targeting PRLR increases tamoxifen sensitivity and enhances the efficacy of chemotherapy in breast cancer

BackgroundThough tamoxifen achieves success in treating estrogen receptor α (ERα)-positive breast cancer, the followed development of tamoxifen resistance is a common challenge in clinic. Signals downstream of prolactin receptor (PRLR) could synergize with ERα in breast cancer progression. However, the potential effect of targeting PRL-PRLR axis combined with tamoxifen has not been thoroughly investigated.MethodsHigh-throughput RNA-seq data obtained from TCGA, Metabric and GEO datasets were analyzed to explore PRLR expression in breast cancer cell and the association of PRLR expression with tamoxifen treatment. Exogenous or PRL overexpression cell models were employed to investigate the role of activated PRLR pathway in mediating tamoxifen insensitivity. Immunotoxin targeting PRLR (N8-PE24) was constructed with splicing-intein technique, and the efficacy of N8-PE24 against breast cancer was evaluated using in vitro and in vivo methods, including analysis of cells growth or apoptosis, 3D spheroids culture, and animal xenografts.ResultsPRLR pathway activated by PRL could significantly decrease sensitivity of ERα-positive breast cancer cells to tamoxifen. Tamoxifen treatment upregulated transcription of PRLR and could induce significant accumulation of PRLR protein in breast cancer cells by alkalizing lysosomes. Meanwhile, tamoxifen-resistant MCF7 achieved by long-term tamoxifen pressure exhibited both upregulated transcription and protein level of PRLR. Immunotoxin N8-PE24 enhanced sensitivity of breast cancer cells to tamoxifen both in vitro and in vivo. In xenograft models, N8-PE24 significantly enhanced the efficacy of tamoxifen and paclitaxel when treating PRLR-positive triple-negative breast cancer.ConclusionsPRL-PRLR axis potentially associates with tamoxifen insensitivity in ERα-positive breast cancer cells. N8-PE24 could inhibit cell growth of the breast cancers and promote drug sensitivity of PRLR-positive breast cancer cells to tamoxifen and paclitaxel. Our study provides a new perspective for targeting PRLR to treat breast cancer.

Abstract PO1-24-06: Prolactin Hormone-Induced Mammary Differentiation and Anti-tumorigenic Role in Breast Cancer

Abstract The premise of differentiation therapy in cancer is a strategy that aims at engaging-forward differentiation and cellular reprograming restricting the proliferative, tumor repopulation, stemness, EMT and metastatic capacities of tumor cells leading to the cessation of the aggressive tumor phenotype and offering the cancer patients improved survival for decades. Therefore, deciphering molecular mechanisms deriving differentiation in normal mammary and breast cancer cells may allow the discovery of innovative differentiation-based biomarkers and therapeutics in breast cancer. Lactation is an intricate process that results in the ability of the breast cells to produce a complex nutritious biological fluid to nurse the new-born. While the beneficial effects of breastfeeding to the infant is irrefutable, its effects on maternal health are less investigated especially in relation to breast cancer that still affects 1 in 8 women in high income countries and 1 in 20 globally. Importantly, epidemiological studies have linked breastfeeding to reduced risk of breast cancer by promoting terminal differentiation of the breast epithelial cells. While breastfeeding process is regulated by multiple hormones, growth factors, and transcriptional regulators, the lactation hormone prolactin (PRL) is known to be directly implicated in the hormonal control of breastfeeding. Extensive research including our own work has shown that PRL/PRL receptor (PRLR) pathway derives mammary gland development and importantly mammary epithelial cell terminal differentiation allowing successful lactation. In contrast the role of PRL in breast cancer is still to be fully defined. Importantly, we have accumulated compelling evidence using in-silico publicly available patient data sets and molecular data implicating this pathway as a clinically relevant pro-differentiation pathway driving differentiation and cellular reprograming suppressing stemness, EMT, metastasis and tumorigenesis in breast cancer. To better characterize PRL signaling mediating its pro-differentiation effects, we used large-scale unbiased proteomics analysis of PRLR/interacting proteins in breast cancer cells. Our analyses revealed several novel PRLR-interactors. We have further confirmed these interactions through co-immunoprecipitations/western blotting analyses in breast cancer cells representative the different breast cancer subtypes. We also examined their functional contributions to mammary differentiation and tumorigenesis using in vitro and in vivo assays. Moreover, we defined their potential clinical relevance using bioinformatics in silico databases of breast cancer. Together our study delineates novel PRL/PRLR-downstream signaling mediators important for mammary differentiation and tumorigenesis and may thus be useful as biomarkers and/or therapeutic targets in breast cancer. Citation Format: Dana Hamam, Suhad Ali. Prolactin Hormone-Induced Mammary Differentiation and Anti-tumorigenic Role in Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-06.

Abstract 4378: Identification of novel TXNIP-dependent antitumor pathways in breast cancer

Abstract Thioredoxin-interacting protein (TXNIP) plays a critical role in glucose metabolism and redox signaling. TXNIP has recently emerged as a potent tumor suppressor in various cancer models. In previous studies, we found TXNIP was activated by UNC0642, an inhibitor of histone methyltransferase, in MDA-MB-231 breast cancer cells. TXNIP activation was coupled with significant suppression of cell proliferation and tumor growth. In contrast, TXNIP knockdown increased MDA-MB-231 cell proliferation and in tumor growth and metastasis in vivo. TXNIP-reconstitution in TXNIP-deficient HCC1954 cells reduced cell proliferation and migration coupled with increased reactive oxygen species production. TXNIP-reconstitution also significantly decreased mitochondrial respiration, mitochondrial membrane potential and glycolysis in HCC1954 cells. To understand the mechanisms underlying TXNIP’s antitumor activity, we performed co-immunoprecipitation and proteomic analyses to uncover TXNIP-interacting proteins. We identified calpastatin (CAST) as a novel TXNIP-interacting protein in breast cancer cells. CAST is an endogenous inhibitor of calpains, a family of calcium-activated cysteine proteases. Intriguingly, CAST association with TXNIP was readily detectable in TXNIP-low, but not in TXNIP-high breast cancer cells. The disassociation of CAST from TXNIP in TXNIP-high cells might allow CAST to inhibit calpain activity to augment TXNIP’s antitumor function. Through RNAseq analysis, we identified a positive correlation between TXNIP expression and IL-24 activation in various breast cancer cells, which were also confirmed by western blotting. IL-24 has emerged as an important cancer therapy target because of its role in promoting cancer-specific apoptosis. Our data also demonstrated that TXNIP upregulated the level of IL-24 and downregulated STAT-3. Moreover, TXNIP exerts multifaceted effects on BC development through the IL24-STAT3 signaling axis, deepening our understanding of its complex role in the disease. Taken together, these studies identified novel TNXIP-dependent pathways and their potential new mechanisms in breast cancer pathogenesis with significant clinical implications. Citation Format: Jasvinder Singh, Bindeshwar Sah, Robert Clarke, Liang Liu. Identification of novel TXNIP-dependent antitumor pathways in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4378.

UNRAVELLING THE INTERACTION BETWEEN GARCINISIDONE-A AND HER2 PROTEIN IN BREAST CANCER: A COMPUTATIONAL STUDY

Objective: One substance found in the leaves of Garcinia cowa Roxb that has anticancer properties is garcinisidone-A. The study aims to simulate the docking of garcinisidone-A (Gar-A), molecular dynamics, and predict the ADME by predicting the binding of the HER2 protein in breast cancer cells and developing new drug candidate options for cancer treatment, often starting with computational analysis.
 Methods: The research method involves computational utilization of pkCSM applications, Gar-A docking simulation with the HER2 protein using Gnina software version 1.0.2, and molecular dynamics conducted with GROMACS 2022.2 and CHARMMGUI applications.
 Results: Gar-A has a molecular weight of less than 500, a Log P value of greater than 5, a limited amount of water solubility, a low level of skin permeability, good intestinal permeability, and a Convolutional Neural Network (CNN) pose score on the HER2 protein of 0.6178. It also does not readily cross the blood-brain barrier, and total clearance values indicate rapid elimination via other excretory routes or enzyme metabolism. Gar-A is thought to have interactions with HER2. There are hydrogen bond interactions with amino acids Lys753 and Asp863, carbon-hydrogen bonds with amino acids Leu785, Ser783, Thr862, and alkyl bonds with amino acids Leu726, Leu852, and Ile767. The stability of the Gar-A-substrate interaction could have been more evident during 100 ns molecular dynamics simulation.
 Conclusion: The physicochemical properties of Gar-A align with Lipinski's rule for drug candidates. ADME predictions indicate good intestinal permeability for Gar-A; however, it suggests it cannot penetrate the blood-brain barrier. The docking results reveal that Gar-A has a value close to one which indicates similar action to its natural ligand and molecular dynamics simulations that Gar-A is less stable. The results illustrate that Gar-A has the potential as a breast anticancer.

The androgen receptor interacts with GATA3 to transcriptionally regulate a luminal epithelial cell phenotype in breast cancer

BackgroundThe androgen receptor (AR) is a tumor suppressor in estrogen receptor (ER) positive breast cancer, a role sustained in some ER negative breast cancers. Key factors dictating AR genomic activity in a breast context are largely unknown. Herein, we employ an unbiased chromatin immunoprecipitation-based proteomic technique to identify endogenous AR interacting co-regulatory proteins in ER positive and negative models of breast cancer to gain new insight into mechanisms of AR signaling in this disease.ResultsThe DNA-binding factor GATA3 is identified and validated as a novel AR interacting protein in breast cancer cells irrespective of ER status. AR activation by the natural ligand 5α-dihydrotestosterone (DHT) increases nuclear AR-GATA3 interactions, resulting in AR-dependent enrichment of GATA3 chromatin binding at a sub-set of genomic loci. Silencing GATA3 reduces but does not prevent AR DNA binding and transactivation of genes associated with AR/GATA3 co-occupied loci, indicating a co-regulatory role for GATA3 in AR signaling. DHT-induced AR/GATA3 binding coincides with upregulation of luminal differentiation genes, including EHF and KDM4B, established master regulators of a breast epithelial cell lineage. These findings are validated in a patient-derived xenograft model of breast cancer. Interaction between AR and GATA3 is also associated with AR-mediated growth inhibition in ER positive and ER negative breast cancer.ConclusionsAR and GATA3 interact to transcriptionally regulate luminal epithelial cell differentiation in breast cancer regardless of ER status. This interaction facilitates the tumor suppressor function of AR and mechanistically explains why AR expression is associated with less proliferative, more differentiated breast tumors and better overall survival in breast cancer.

Targeting AR-positive breast cancer cells via drug repurposing approach

Androgen Receptor (AR) is overexpressed in almost all the molecular subtypes of breast cancer. Besides aiding the tumorigenic environment of cancer by abnormal cell proliferation, AR also takes part in promoting cancer signaling pathways, thereby promoting aggressiveness. In this study, AR was selected as the target protein in breast cancer cells. Following this, a library of 1293 FDA-approved drugs was screened via molecular docking, MD simulation, and MMPBSA binding energy. Amongst the library of compounds, Adapalene exhibited the least binding energy of (−10.2 kCal/mol) in comparison to that of the chosen reference compound, Nilutamide (−8.6 kCal/mol). Furthermore, the in vitro efficacy of Adapalene was also determined in two different breast cancer cell lines such as MCF7 (AR-positive/ER-positive) and MDA-MB-231 (AR negative/TNBC). Initially, the cell viability assay (MTT) was performed, which endowed us with a lesser IC50 value of Adapalene in comparison to Nilutamide in both cell lines. The IC50 of Adapalene was found to be 12 μM and 39.4 μM in MCF7 and MDA-MB-231 cells, respectively. Furthermore, Adapalene also induced cellular ROS and apoptosis by 3.5-fold and 26.58% in MCF7 cells. However, the overall effect of Adapalene was significantly lower in the case of MDA-MB-231 cell lines, which could be attributed to its inherent nature of the absence of hormone receptors. Conclusively, Adapalene possesses greater therapeutic efficacy in comparison to the control drug, thereby hinting towards the potential use of Adapalene in the treatment of AR-positive breast cancer.

PKCζ phosphorylates VASP to mediate chemotaxis in breast cancer cells

Breast carcinoma (BC) is one of the most common malignant cancers in females, and metastasis remains the leading cause of death in these patients. Chemotaxis plays an important role in cancer cell metastasis and the mechanism of breast cancer chemotaxis has become a central issue in contemporary research. PKCζ, a member of the atypical PKC family, has been reported to be an essential component of the EGF-stimulated chemotactic signaling pathway. However, the molecular mechanism through which PKCζ regulates chemotaxis remains unclear. Here, we used a proteomic approach to identify PKCζ-interacting proteins in breast cancer cells and identified VASP as a potential binding partner. Intriguingly, stimulation with EGF enhanced this interaction and induced the translocalization of PKCζ and VASP to the cell membrane. Further experiments showed that PKCζ catalyzes the phosphorylation of VASP at Ser157, which is critical for the biological function of VASP in regulating chemotaxis and actin polymerization in breast cancer cells. Furthermore, in PKCζ knockdown BC cells, the enrichment of VASP at the leading edge was reduced, and its interaction with profilin1 was attenuated, thereby reducing the chemotaxis and overall motility of breast cancer cells after EGF treatment. In functional assays, PKCζ promoted chemotaxis and motility of BC cells through VASP. Our findings demonstrate that PKCζ, a new kinase of VASP, plays an important role in promoting breast cancer metastasis and provides a theoretical basis for expanding new approaches to tumor biotherapy.

Non-POU Domain-Containing Octomer-Binding (NONO) protein expression and stability promotes the tumorigenicity and activation of Akt/MAPK/β-catenin pathways in human breast cancer cells

Breast cancer is one of the most common cancers with a high mortality rate, underscoring the need to identify new therapeutic targets. Here we report that non-POU domain-containing octamer-binding (NONO) protein is overexpressed in breast cancer and validated the interaction of the WW domain of PIN1 with c-terminal threonine-proline (thr-pro) motifs of NONO. The interaction of NONO with PIN1 increases the stability of NONO by inhibiting its proteasomal degradation, and this identifies PIN1 as a positive regulator of NONO in promoting breast tumor development. Functionally, silencing of NONO inhibits the growth, survival, migration, invasion, epithelial to mesenchymal transition (EMT), and stemness of breast cancer cells in vitro. A human metastatic breast cancer cell xenograft was established in transparent zebrafish (Danio rerio) embryos to study the metastatic inability of NONO-silenced breast cancer cells in vivo. Mechanistically, NONO depletion promotes the expression of the PDL1 cell-surface protein in breast cancer cells. The identification of novel interactions of NONO with c-Jun and β-catenin proteins and activation of the Akt/MAPK/β-catenin signaling suggests that NONO is a novel regulator of Akt/MAPK/β-catenin signaling pathways. Taken together, our results indicated an essential role of NONO in the tumorigenicity of breast cancer and could be a potential target for anti-cancerous drugs.C-mep14XPctyTeLC54ZqwrVideo

KCNJ15 deficiency promotes drug resistance via affecting the function of lysosomes

The altered lysosomal function can induce drug redistribution which leads to drug resistance and poor prognosis for cancer patients. V-ATPase, an ATP-driven proton pump positioned at lysosomal surfaces, is responsible for maintaining the stability of lysosome. Herein, we reported that the potassium voltage-gated channel subfamily J member 15 (KCNJ15) protein, which may bind to V-ATPase, can regulate the function of lysosome. The deficiency of KCNJ15 protein in breast cancer cells led to drug aggregation as well as reduction of drug efficacy. The application of the V-ATPase inhibitor could inhibit the binding between KCNJ15 and V-ATPase, contributing to the amelioration of drug resistance. Clinical data analysis revealed that KCNJ15 deficiency was associated with higher histological grading, advanced stages, more metastases of lymph nodes, and shorter disease free survival of patients with breast cancer. KCNJ15 expression level is positively correlated with a high response rate after receiving neoadjuvant chemotherapy. Moreover, we revealed that the small molecule drug CMA/BAF can reverse drug resistance by disrupting the interaction between KCNJ15 and lysosomes. In conclusion, KCNJ15 could be identified as an underlying indicator for drug resistance and survival of breast cancer, which might guide the choice of therapeutic strategies.

Anti-cancer Effect of Unique Cartilage Matrix-associated Protein in Breast Cancer Cells Depends on γ-Carboxylation.

Unique cartilage matrix-associated protein (UCMA), a recently discovered vitamin K-dependent protein (VKDP) with a large number of γ-carboxyglutamic acid (Gla) residues, is associated with ectopic calcifications. Although the function of VKDPs is related to their γ-carboxylation status, the carboxylation status of UCMA in breast cancer is still unknown. Here, we investigated the inhibitory effect of UCMA with differing γ-carboxylation status on breast cancer cell lines, such as MDA-MB-231, 4T1, and E0771 cells. Undercarboxylated UCMA (ucUCMA) was generated by mutating the γ-glutamyl carboxylase (GGCX) recognition sites. The ucUCMA and carboxylated UCMA (cUCMA) proteins were collected from culture media of HEK293-FT cells that had been transfected with mutated GGCX and wild-type UCMA expression plasmids, respectively. Boyden Transwell and colony formation assays were performed to evaluate cancer cell migration, invasion, and proliferation. Culture medium containing cUCMA protein inhibited the migration, invasion, and colony formation of MDA-MB-231 and 4T1 cells to a greater degree than medium containing ucUCMA protein. Significant reductions in the migration, invasion, and colony formation were also observed in cUCMA-treated E0771 cells compared to those in ucUCMA-treated cells. The inhibitory role of UCMA in breast cancer is closely related to its γ-carboxylation status. The results of this study may be a basis for the development of UCMA-based anti-cancer drugs.

Codon adaptation by synonymous mutations impacts the functional properties of the estrogen receptor-alpha protein in breast cancer cells.

Oestrogen receptor-alpha (ERα) positivity is intimately associated with the development of hormone-dependent breast cancers. A major challenge in the treatment of these cancers is to understand and overcome the mechanisms of endocrine resistance. Recently, two distinct translation programmes using specific transfer RNA (tRNA) repertoires and codon usage frequencies were evidenced during cell proliferation and differentiation. Considering the phenotype switch of cancer cells to more proliferating and less-differentiated states, we can speculate that the changes in the tRNA pool and codon usage that likely occur make the ERα coding sequence no longer adapted, impacting translational rate, co-translational folding and the resulting functional properties of the protein. To verify this hypothesis, we generated an ERα synonymous coding sequence whose codon usage was optimized to the frequencies observed in genes expressed specifically in proliferating cells and then investigated the functional properties of the encoded receptor. We demonstrate that such a codon adaptation restores ERα activities to levels observed in differentiated cells, including: (a) an enhanced contribution exerted by transactivation function 1 (AF1) in ERα transcriptional activity; (b) enhanced interactions with nuclear receptor corepressor 1 and 2 [NCoR1 and NCoR2 (also known as SMRT) respectively], promoting repressive capability; and (c) reduced interactions with SRC proto-oncogene, non-receptor tyrosine kinase (Src) and phosphoinositide 3-kinase (PI3K) p85 kinases, inhibiting MAPK and AKT signalling pathway.

E2F1-initiated transcription of PRSS22 promotes breast cancer metastasis by cleaving ANXA1 and activating FPR2/ERK signaling pathway

Breast cancer (BC) is the most common malignant tumor in women worldwide. Metastasis is the main cause of BC-related death. The specific mechanism underlying BC metastasis remains obscure. Recently, PRSS22 was discovered to be involved in tumor development, however, its detailed biological function and regulatory mechanism in BC are unclear. Here, we characterized that PRSS22 expression is upregulated in BC tissues compared with non-tumorous breast tissues. Dual luciferase assays, bioinformatics analyses and chromatin immunoprecipitation (ChIP) assays indicated that transcription factor E2F1 directly binds to the PRSS22 promoter region and activates its transcription. Functionally, upregulation of PRSS22 promoted invasion and metastasis of BC cells in vitro and in vivo, whereas knockdown of PRSS22 inhibited its function. Mechanistically, the combination of PRSS22 and ANXA1 protein in BC cells was first screened by protein mass spectrometry analysis, and then confirmed by co-immunoprecipitation (Co-IP) and western blot assays. Co-overexpression of PRSS22 and ANXA1 could promote BC cell migration and invasion. We further demonstrated that PRSS22 promotes the cleavage of ANXA1 and in turn generates an N-terminal peptide, which initiates the FPR2/ERK signaling axis to increase BC aggressiveness.

Bovine holo-lactoferrin inhibits migration and invasion in MDA-MB-231 breast cancer cells.

Breast cancer is the most common malignancy in developed countries and the main cause of deaths in women worldwide. Lactoferrin (Lf) is an iron-binding protein constituted for a single polypeptide chain that is folded into two symmetrical lobes that bind Fe2+ or Fe3+. Lf has the ability to reversibly bind Fe3+ and is found free of Fe3+ (Apo-Lf) or associated with Fe3+ (Holo-Lf) with a different three-dimensional conformation. However, the role of bovine Apo-Lf (Apo-BLf) and bovine Holo-Lf (Holo-BLf) in the migration and invasion induced by linoleic acid (LA) and fetal bovine serum (FBS), as well as in the expression of mesenchymal and epithelial proteins in breast cancer cells has not been studied. Scratch wound assays demonstrated that Holo-BLf and Apo-BLf do not induce migration, however they differentially inhibit the migration induced by FBS and LA in breast cancer cells MDA-MB-231. Western blot, invasion, zymography and immunofluorescence confocal microscopy assays demonstrated that Holo-BLf partly inhibit the invasion, FAK phosphorylation at tyrosine (Tyr)-397 and MMP-9 secretion, whereas it increased the number and size of focal adhesions induced by FBS in MDA-MB-231 cells. Moreover, Holo-BLf induced a slight increase of E-cadherin expression in MCF-7 cells, and inhibited vimentin expression in MCF-7 and MDA-MB-231 breast cancer cells. Holo-BLf inhibits cellular processes that mediate the invasion process in breast cancer cells.

Impact of cancer stem cell markers Nestin and Sox2 on cell migration of epithelial and mesenchymal pancreatic cancer cells

Breast cancer (BC) is common cancer in female globally. Sevoflurane (SEV) has been reported to inhibit the metastasis of multiple cancers, including glioma, colorectal cancer, and hepatocellular carcinoma. However, the role of SEV in the metastasis of BC cells remains poorly understood.Transwell migration and invasion assays were performed to detect the migration and invasion of BC cells. Western blot assay was carried out to measure epithelial-mesenchymal transition (EMT)-related proteins in BC cells, including E-cadherin, N-cadherin, and fibronectin. Quantitative real-time polymerase chain reaction was conducted to determine the enrichment of miR-139-5p and ADP-ribosylation factor 6 (ARF6) in BC tissues and cells. The protein expression of ARF6 in BC tissues and cells was measured by western blot assay. The target of miR-139-5p was predicted by starBase software, and the target relationship between miR-139-5p and ARF6 in BC cells was confirmed by dual-luciferase reporter assay.SEV suppressed the migration, invasion, and EMT of BC cells, especially in the high-concentration SEV group. The level of miR-139-5p was lower in BC tissues and cells than that in paired normal tissues and normal mammary epithelial cells MCF-10A. MiR-139-5p was upregulated in BC cells treated with SEV. ARF6 was upregulated in BC tissues and cells compared with that in corresponding normal tissues and normal mammary epithelial cells MCF-10A. SEV reduced the mRNA and protein expression of ARF6 in BC cells. The accumulation of ARF6 or the interference of miR-139-5p reversed the suppressive effects of SEV treatment on the migration, invasion, and EMT of BC cells. MiR-139-5p bound to ARF6 and inversely modulated the level of ARF6 in BC cells. The transfection of si-ARF6 attenuated the promoting effects of miR-139-5p depletion on the migration, invasion, and EMT of BC cells treated with SEV.SEV suppressed the migration, invasion, and EMT of BC cells through downregulating the abundance of ARF6 by upregulating miR-139-5p. The miR-139-5p/ARF6 axis might be a promising target for the treatment of BC.

PER3 plays anticancer roles in the oncogenesis and progression of breast cancer via regulating MEK/ERK signaling pathway.

The study aimed at exploring the expression of period circadian regulator 3 (PER3), a major member of the circadian clock gene family, and its biological function in breast cancer. PER3-silencing and PER3-overexpression cell lines were established by transfecting with pGenesil1-PER3 and Lenti-blast-PER3 vector, respectively. The results showed that the expression of PER3 was downregulated in breast cancer tissues and cell lines ( p < 0.001), and its low expression was significantly correlated with advanced tumor stage ( p = 0.031) and advanced T stage ( p = 0.018). Cell functional experiments indicated that the silencing of PER3 elevated the ability of breast cancer cells to proliferate, invade, and metastasize in vitro ( p < 0.05), whereas overexpression of PER3 had an inhibitory effect on these malignant phenotype of breast cancer cells ( p < 0.05). Moreover, the activation of MEK/ERK signaling pathway was evidently inhibited by silencing of PER3, as evidenced by decreased expression levels of p-MEK and p-ERK1/2 proteins in breast cancer cells ( p < 0.05). PER3-silencing and PER3-overexpression cells were treated with PD98059 (an inhibitor of MEK/ERK signaling) and TPA (an activator of MEK/ERK signaling), respectively. It was observed that PER3 silencing-mediated malignant phenotype in breast cancer cells was markedly suppressed by PD98059 treatment. Instead, TPA exposure reversed the inhibitory effects of PER3 overexpression on DNA synthesis, proliferation, migration, and invasion of breast cancer cells. These findings suggested that PER3 function as a tumor suppressor in the development and progression of breast cancer and its anticancer roles might be dependent on the MEK/ERK signaling pathway.

Up-regulation of CPNE1 Appears to Enhance Cancer Progression in HER2-positive and Luminal A Breast Cancer Cells.

Copine 1 (CPNE1) is a calciumdependent phospholipid protein that has been shown to regulate the AKT serine/threonine kinase 1 (AKT) signaling pathway to mediate its function in various cell types. However, little is known about the physiological function of this protein in breast cancer cells. We aimed to investigate the prognostic and therapeutic value of CPNE1 in erb-b2 receptor tyrosine kinase 2 [human epidermal growth factor receptor 2 (HER2)]-positive and luminal A subtypes of breast cancer. Western blotting, cell viability, wound-healing and invasion assays were performed on SK-BR3 and MCF-7 breast cancer cells with forced overexpression of CPNE1. CPNE1 immunohistochemical (IHC) staining and bioinformatics analysis were performed on specimens from patients with breast cancer and compared to normal breast samples. CPNE1 overexpression promoted AKT activation, and increased cell viability and cell motility in SK-BR3 and MCF-7 breast cancer cells. In addition, invasive capabilities of SK-BR3 cells were increased by the overexpression of CPNE1. The expression levels of CPNE1 were higher in HER2-positive and luminal A subtypes of human breast cancer tissues compared with those in adjacent normal tissues. Furthermore, CPNE1 expression was increased in RNA microarray analysis of samples from patients with breast cancer compared to normal breast samples. CPNE1 may play a key role in the pathophysiology of HER2-positive and luminal A subtypes of breast cancer.

Multifaceted Roles of the E3 Ubiquitin Ligase RING Finger Protein 115 in Immunity and Diseases.

Ubiquitination is a post-translational modification that plays essential roles in various physiological and pathological processes. Protein ubiquitination depends on E3 ubiquitin ligases that catalyze the conjugation of ubiquitin molecules on lysine residues of targeted substrates. RING finger protein 115 (RNF115), also known as breast cancer associated gene 2 (BCA2) and Rab7-interacting RING finger protein (Rabring7), has been identified as a highly expressed protein in breast cancer cells and tissues. Later, it has been demonstrated that RNF115 catalyzes ubiquitination of a series of proteins to modulate a number of signaling pathways, and thereby regulates viral infections, autoimmunity, cell proliferation and death and tumorigenesis. In this review, we introduce the identification, expression and activity regulation of RNF115, summarize the substrates and functions of RNF115 in different pathways, and discuss the roles of RNF115 as a biomarker or therapeutic target in diseases.