Regulates Breast Cancer Cell Proliferation Research Articles

Membrane trafficking alterations in breast cancer progression.

Breast cancer (BC) is the most common type of cancer in women, and remains one of the major causes of death in women worldwide. It is now well established that alterations in membrane trafficking are implicated in BC progression. Indeed, membrane trafficking pathways regulate BC cell proliferation, migration, invasion, and metastasis. The 22 members of the ADP-ribosylation factor (ARF) and the >60 members of the rat sarcoma (RAS)-related in brain (RAB) families of small GTP-binding proteins (GTPases), which belong to the RAS superfamily, are master regulators of membrane trafficking pathways. ARF-like (ARL) subfamily members are involved in various processes, including vesicle budding and cargo selection. Moreover, ARFs regulate cytoskeleton organization and signal transduction. RABs are key regulators of all steps of membrane trafficking. Interestingly, the activity and/or expression of some of these proteins is found dysregulated in BC. Here, we review how the processes regulated by ARFs and RABs are subverted in BC, including secretion/exocytosis, endocytosis/recycling, autophagy/lysosome trafficking, cytoskeleton dynamics, integrin-mediated signaling, among others. Thus, we provide a comprehensive overview of the roles played by ARF and RAB family members, as well as their regulators in BC progression, aiming to lay the foundation for future research in this field. This research should focus on further dissecting the molecular mechanisms regulated by ARFs and RABs that are subverted in BC, and exploring their use as therapeutic targets or prognostic markers.

Functional Roles of Tumor Protein D52 (TPD52) in Breast Cancer

Abstract: Breast cancer is an aggressive disease with a significant morbidity and death rate among women worldwide. Despite the progress of diagnostic and therapy options for breast cancer in recent years, the prognosis and survival rates of breast cancer patients remain unsatisfactory. The aberrant growth and spread of tumor cells are the leading cause of death in these patients. More profound knowledge of molecular biology underlying breast cancer and a more accurate stratification are still necessary for more precise therapy. Further understanding of the disease's molecular mechanism and genetic aberrations may allow for the identification of more accurate prognostic and diagnostic markers and more effective treatments. Tumor protein D52 (TPD52) is an oncogene whose overexpression has been found in breast cancer. Overexpression of TPD52 has been linked to specific molecular subtypes of breast cancer, including luminal B and ERBB2-positive tumors. Besides, non-coding RNAs (ncRNAs) were found to play a significant role in breast cancer progression. ncRNAs play regulatory roles in cell behaviors, cancer pathogenesis, radiotherapy, and resistance to chemotherapy. Multiple ncRNAs could modulate the expression of TPD52 and regulate breast cancer cell proliferation, invasion, and metastasis. In this review, we summarized the functions of TPD52 in breast cancer cells.

TRPV3 regulates Breast Cancer Cell Proliferation and Apoptosis by EGFR/AKT pathway.

Breast cancer (BC) is one of the most common cancer types worldwide and the first cause of cancer-related deaths in women. Transient receptor potential vanillin 3 (TRPV3) has been preliminarily discovered to play an important role in various cancers, including BC. Here, we explored the effect of TRPV3 on breast cancer cells and its potential mechanism. TRPV3 level was measured in BC tissue and adjacent noncancerous breast tissue using real-time RT-PCR and Western blot. Wound healing was used to detect cell migration. MTT and EDU were detected cell proliferation. TUNEL and Caspase-3 activity were used to detect cell apoptosis. We found that TRPV3 expression significantly increased in both human BC tissues and breast cells line. TRPV3 siRNA (TRPV3 inhibition) dramatically suppressed cell migration and proliferation, promoted the apoptosis, and decreased [Ca2+]i; whereas Carvacrol (TRPV3 agonist) has opposite effect in MCF-7 cells. We validated EGFR (Epidermal growth factor receptor) is a direct target protein of TRPV3. Mechanism studies have shown that Carvacrol increased phosphorylation levels of EGFR and AKT, and were decreased by suppression of TRPV3. Moreover, Erlotinib (EGFR inhibitor) and LY294002 (PI3K inhibitor) diminished Carvacrol induced cell migration and proliferation, promoted cell apoptosis, and increased [Ca2+]i in Carvacrol group. Our results collectively suggest that TRPV3 siRNA inhibits migration and proliferation, and promoted apoptosis in breast cancer cells by EGFR/AKT pathway. These findings indicate that TRPV3 may represent a novel therapeutic strategy for breast cancer.

Low-Density Lipoprotein Contributes to Endometrial Carcinoma Cell Proliferation, Migration, and Invasion by Activating the JAK-STAT Signaling Pathway.

Cholesterol-rich low-density lipoprotein (LDL) particles have been demonstrated to regulate breast cancer cell proliferation and migration, but their biological function and relevant mechanisms in endometrial carcinoma (EC) remain unclear. Serum and tissue samples were collected from EC patients (n = 50) and patients with benign endometrial hyperplasia (n = 50). Ishikawa and RL95-2 cells were stimulated with different concentrations of LDL, followed by treatment with a JAK2 inhibitor (SD-1029). LDL concentrations were determined by ELISA. The in vitro biological behavior of cells was examined using the CCK-8 assay, EdU staining, and Transwell assay. The tumorigenicity of LDL in vivo was examined using a xenograft mouse model. western blotting, immunofluorescence, and immunohistochemistry studies were performed to measure related protein expression. The LDL concentrations and levels of p-JAK2 and p-STAT3 expression were elevated in the clinical samples. Similar trends in expression were detected in EC cells after LDL stimulation. LDL treatment significantly promoted EC cell proliferation, migration, and invasion, and also upregulated p-JAK2 and p-STAT3 expression in a dose-dependent manner. Moreover, SD-1029 dramatically blocked the LDL-mediated effects on EC cells. Intravenous injection of LDLs promoted tumor growth in the xenograft nude mice, and also increased p-JAK2, p-STAT3, and Ki-67 expression, and downregulated caspase-3 expression. These findings indicate that LDLs exert an oncogenic effect in EC cells by activating the JAK/STAT signaling pathway, and also suggest the JAK/STAT pathway as a possible therapeutic target for EC.

Regulation of Breast Cancer Cell Proliferation and Immunity via Targeting Autophagic Process Using siRNA Antagonist Atg-5 in MCF-7 Cells.

Background: Breast cancer initiates when normal breast epithelial cells begin to replicate unusually. These cells divide more rapidly than normal cells and continue to be accumulated and formed a lump or mass. Cells may migrate (metastasis stage) from breast tissues to lymph nodes or other human body parts. Cellular autophagy is one of the most critical mechanisms in breast cancer development. Autophagy plays a dual function as a tumor support process or suppressor mechanism. Accordingly, we aimed to highlight the molecular role of autophagy in breast cancer development and cancer therapy using RNA interference (RNAi). 
 Material and Methods: MCF-7 cell line was used to investigate the direct connection between breast cancer and the autophagy pathway using a respective siRNA antagonist, Atg-5. 
 Results: MCF-7 cell morphology was markedly influenced by transfection with siRNA antagonist Atg-5 compared with control-transfected and untreated cells. Likewise, the number of liPleving MCF-7 cells reduced in transfected cells with siRNA that antagonist ATG-5, while the relative lactate dehydrogenates (LDH) production significantly increased compared with control-transfected cells. Notably, the knockdown efficiency of Atg-5 gene expression was almost 60% inhibition when the MCF-7 was transfected with the siRNA antagonist Atg-5. Furthermore, the relative gene expression of interleukin -8 (IL-8) and transforming growth factor-beta (TGF-β) downregulated in response to depleted Atg-5 in transfected cells. 
 Conclusion: Our findings demonstrate that downregulation of Atg-5 leads to sufficient disturbance of autophagic machinery and subsequently regulates breast cancer cell proliferation.

YBX1/lncRNA SBF2-AS1 interaction regulates proliferation and tamoxifen sensitivity via PI3K/AKT/MTOR signaling in breast cancer cells.

Y-box binding protein 1 (YBX1) is a multifunctional oncoprotein that can interact with several long non-coding RNAs (lncRNAs)to regulate metastasis in malignancies including breast cancer (BC). In the present study, we demonstrated the association of YBX1 with oncogenic lncRNA SBF2-AS1 (SET-binding factor 2 antisense RNA 1) via PI3K/AKT/mTOR signaling to regulate BC cell proliferation. We further explored the involvement of the YBX1/SBF2-AS1/PI3K/AKT/mTOR axis in the restoration of tamoxifen (TAM) sensitivity. YBX1-SBF2-AS1 association was predicted in silico and verified by RNA immunoprecipitation (RIP)-qPCR assay. Transfection experiments, Real-time RT PCR, Western blots, Phospho AKT/mTOR antibody array kit, and cell proliferation/apoptosis assays were employed to detect the YBX1/SBF2-AS1/ PI3K/AKT/mTOR axis and its effects upon TAM treatment in vitro. We identified that the YBX1 protein specifically binds to lncRNA SBF2-AS1. Our transfection experiments in MCF-7 and MDA-MB-468 cells with SBF2-AS1 silenced or overexpressed YBX1 plasmids, and their negative controls revealed that YBX1 regulates the expression of SBF2-AS1 by forming a positive feedback loop for its activation. We further demonstrated YBX1-SBF2-AS1 association exerts its effects on cell proliferation via PI3K/AKT/mTOR signaling pathway. Furthermore, we observed an increase in TAM sensitivity in BC cells after the knockdown of YBX1-SBF2-AS1 marked by decreased cell proliferation through disruption of the PI3K/AKT/mTOR axis. Our study has identified a novel YBX1/SBF2-AS1/PI3K/AKT/mTOR regulatory axis which may serve as a potential target to improve the effectiveness and efficacy of TAM treatment in BC.

RAB5C, a new mRNA binding target of HuR, regulates breast cancer cell proliferation.

The posttranscriptional control of gene expression mediated by RNA-binding proteins (RBPs) is essential to determine tumor cell fate. HuR is an RBP with increased expression in various cancer types. This study aimed to clarify the regulatory mechanism of HuR's contribution to breast cancer (BC) cell proliferation by inducing RAB5C expression. First, we analyzed the expression level of HuR and RAB5C in BCtissues and cell lines by immunohistochemistry, qRT-PCR, and western blot. Next, to further investigate the effect of HuR on RAB5C expression, we used short hairpin RNAs(shRNAs) to silence endogenous HuR expression in BCcell lines MCF7 and MDA-MB-231. The binding site of RAB5C mRNA and HuR was confirmed by RNA immunoprecipitation. Finally, the function of RAB5C was investigated using flow cytometry, colony formation, and MTT assays. We found that the expression of HuR and RAB5C was significantly upregulated in BC tissues and MCF-7 and MDA-MB231 cell lines. Importantly, RAB5C mRNA stability was increased through binding of HuR to its 3'UTR. Inhibition of HuR expression using shRNA decreased RAB5C mRNA, suggesting that HuR plays a role in regulating RAB5C expression level. In addition, suppression of RAB5C expression reduced BC cell growth. These results suggest RAB5C functions as an oncogene in BCcells, HuR promoted BCcell survival by facilitating RAB5C expression. Our findings suggest that HuR and RAB5C play important roles in BCcell survival.

G3BP1 regulates breast cancer cell proliferation and metastasis by modulating PKCζ.

Breast cancer is a leading cause of death and morbidity among female cancers. Several factors, including hormone levels, lifestyle, and dysregulated RNA-binding proteins, have been associated with the development of breast cancer. Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and protein kinase C, Zeta isoform (PKCζ) are oncogenes implicated in numerous cancers, including breast cancer. However, their interaction and role in promoting breast cancer proliferation and metastasis have not been well-characterized. In the present study, we demonstrated that G3BP1 expression was elevated in breast cancer and that knockdown of G3BP1 diminished the proliferation and metastasis of breast cancer cells. Mechanistically, we identified proliferation and a series of metastasis-related properties, including chemotaxis, migration, Golgi polarity localization, and actin polymerization, that were modulated by G3BP1 knockdown. We found that G3BP1 and PKCζ were co-localized and interacted intracellularly, and they co-underwent membrane translocation under EGF stimulation. Following the knockdown of G3BP1, we observed the membrane translocation and phosphorylation of PKCζ were significantly impaired, suggesting that G3BP1 regulates the activation of PKCζ. Our findings indicate that G3BP1 plays multiple roles in breast cancer cell proliferation and metastasis. The activation of PKCζ by G3BP1 may be the specific mechanism underlying the process.

SEC61G regulates breast cancer cell proliferation and metastasis by affecting the Epithelial-Mesenchymal Transition.

Breast cancer is a common malignant tumor for women and its incidence has increased constantly in recent decades. The underlying molecular means of breast tumorigenesis endure uncertain. With the sequencing expertise, we found that the SEC61G gene is overexpressed in tumor tissues. However, the biological function of SEC61G in breast malignancy has yet to be determined.We investigated the SEC61G expression level, genetic alteration, IHC, immune infiltration, diagnostic value, survival analysis, and functional enrichment analysis by bioinformatics analysis. Then, vitro experiments were done. We investigated that SEC61G was greater in breast cancer tissues related to adjacent non-tumor tissues through qRT-PCR. We performed proliferation, colony formation, migration, invasion assays, and EMT-related phenotype to determine the specific biological functions of SEC61G in breast cancer cell lines (MDA-MB-231, BT-549) transfected with small interfering RNA.SEC61G expression and exon expression were higher in the tumor while the level of SEC61G methylation was higher in normal tissues. The expression level of SEC61G was connected with immune infiltration and survival and was an effective diagnostic and prognostic indicator. The functional enrichment analysis of SEC61G prompted that SEC61G might play a tumor-promoting role via the EMT pathway. In vitro experiments indicated that knocking down SEC61G considerably impaired the colony formation, cck-8, migration, and invasion, and induced apoptosis of the breast cancer cell lines. The vitro experiments also indicated that ectopic expression of SEC61G could influence EMT.This study revealed that SEC61G plays vital tumorigenic functions and acts as a novel oncogene in breast cancer.

Abstract 2573: The anticancer effects of the vitamin E isoform, γ-tocotrienol, and vitamin D3 act synergistically to inhibit MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation and viability in vitro

Abstract Epidemiological studies suggest that dietary intake of some natural products may be effective in reducing the risk of breast cancer. Furthermore, various phytochemicals have been shown to interfere with cell signaling pathways involved in regulating breast cancer cell proliferation and viability. γ-Tocotrienol is a natural isoform of vitamin E that displays potent anticancer activity against a variety of cancer cell types at treatment doses that have little or no effect on normal cell growth or viability. Vitamin D3 is an abundant phytochemical which also displays potent antiproliferative and antiangiogenic activity against human breast cancer cells. Studies were conducted to examine the effects of γ-tocotrienol and vitamin D3 given alone or in combination on MDA-MB-231 triple negative breast cancer (TNBC) cellular proliferation, migration, colony formation, invasion, cell cycle progression, and apoptosis. Results showed that combined treatment with subeffective doses of γ-tocotrienol (8 μM) and vitamin D3 (10 nM) synergistically inhibited the growth of MDA-MB-231 cells, as determined by the MTT assay and isobologram analysis. Additional studies indicated that this combination treatment resulted in a significant inhibition in cancer cell migration and a significant reduction in cancer cell colony formation. Studies using the Matrigel invasion assay showed that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly inhibited MDA-MB-231 invasiveness. Flow cytometry analysis showed that similar combined treatment of γ-tocotrienol and vitamin D3 significantly increased the percentage of cells found in G0/G1 phase, as compared to cells in the vehicle-treated control group. After 4 days of combined treatment with subeffective doses of γ-tocotrienol and vitamin D3, the percentage of cells in the G0/G1 phase of the cell cycle increased to 55% (p<0.05, all p values compared to untreated cells), as compared to 37.48% for cells in the vehicle-treated control group. In addition, combination treatment with subeffective doses of γ-tocotrienol and vitamin D3, significantly increased the number of apoptotic and necrotic cells, as comparted to cells in the vehicle-treated control group. In summary, these findings indicate that combined treatment with subeffective doses of γ-tocotrienol and vitamin D3 act synergistically to inhibit MDA-MB-231 human breast cancer cell growth and viability, and strongly suggest that combined treatment with these agents may provide some benefits in the treatment of TNBC in women. This study was supported in part by funding from the Louisiana Cancer Foundation. Citation Format: Md. R. Anwar, A.K.M.N. Hossian, Georgios Matthaiolampakis, Paul W. Sylvester. The anticancer effects of the vitamin E isoform, γ-tocotrienol, and vitamin D3 act synergistically to inhibit MDA-MB-231 triple negative breast cancer (TNBC) cell proliferation and viability in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2573.

Mutational Landscape of Pirin and Elucidation of the Impact of Most Detrimental Missense Variants That Accelerate the Breast Cancer Pathways: A Computational Modelling Study.

Pirin (PIR) protein is highly conserved in both prokaryotic and eukaryotic organisms. Recently, it has been identified that PIR positively regulates breast cancer cell proliferation, xenograft tumor formation, and metastasis, through an enforced transition of G1/S phase of the cell cycle by upregulation of E2F1 expression at the transcriptional level. Keeping in view the importance of PIR in many crucial cellular processes in humans, we used a variety of computational tools to identify non-synonymous single-nucleotide polymorphisms (SNPs) in the PIR gene that are highly deleterious for the structure and function of PIR protein. Out of 173 SNPs identified in the protein, 119 are non-synonymous, and by consensus, 24 mutations were confirmed to be deleterious in nature. Mutations such as V257A, I28T, and I264S were unveiled as highly destabilizing due to a significant stability fold change on the protein structure. This observation was further established through molecular dynamics (MD) simulation that demonstrated the role of the mutation in protein structure destability and affecting its internal dynamics. The findings of this study are believed to open doors to investigate the biological relevance of the mutations and drugability potential of the protein.

Identification of Nucleolin as a Novel AEG-1-Interacting Protein in Breast Cancer via Interactome Profiling.

Simple SummaryBreast cancer is one of the most common cancers affecting women today. Astrocyte Elevated Gene-1 (AEG-1) is elevated in breast cancer patients and is associated with metastasis and poor prognosis. However, the mechanisms by which AEG-1 promotes breast cancer are not fully understood. This report focuses on a novel AEG-1 interacting protein, nucleolin (NCL), which we identified via mass spectrometry-based interactome profiling. We found NCL to be important for the oncogenic function of AEG-1 in breast cancer. Further, c-Met was identified as a novel mediator of the oncogenic function of the AEG-1-NCL complex. Collectively, our study suggests that targeting the AEG-1-NCL protein complex could be an effective therapeutic approach for the treatment of some breast cancers.Breast cancer is one of the most common malignant diseases worldwide. Astrocyte elevated gene-1 (AEG-1) is upregulated in breast cancer and regulates breast cancer cell proliferation and invasion. However, the molecular mechanisms by which AEG-1 promotes breast cancer have yet to be fully elucidated. In order to delineate the function of AEG-1 in breast cancer development, we mapped the AEG-1 interactome via affinity purification followed by LC-MS/MS. We identified nucleolin (NCL) as a novel AEG-1 interacting protein, and co-immunoprecipitation experiments validated the interaction between AEG-1 and NCL in breast cancer cells. The silencing of NCL markedly reduced not only migration/invasion, but also the proliferation induced by the ectopic expression of AEG-1. Further, we found that the ectopic expression of AEG-1 induced the tyrosine phosphorylation of c-Met, and NCL knockdown markedly reduced this AEG-1 mediated phosphorylation. Taken together, our report identifies NCL as a novel mediator of the oncogenic function of AEG-1, and suggests that c-Met could be associated with the oncogenic function of the AEG-1-NCL complex in the context of breast cancer.

SEC61G promotes breast cancer development and metastasis via modulating glycolysis and is transcriptionally regulated by E2F1

Breast cancer is the most common cancer in women and its incidence rates are rapidly increasing in China. Understanding the molecular mechanisms of breast cancer tumorigenesis enables the development of novel therapeutic strategies. SEC61G is a subunit of the endoplasmic reticulum translocon that plays critical roles in various tumors. We aimed to investigate the expression and function of SEC61G in breast cancer. By analyzing The Cancer Genome Atlas breast cancer cohort, we found that SEC61G was highly expressed in breast cancer and predicted poor prognosis of breast cancer patients. Overexpression of SEC61G and its prognostic role was also confirmed in the Nanjing Medical University (NMU) breast cancer cohort. Functionally, we demonstrated that knockdown of SEC61G suppressed breast cancer cell proliferation, migration, invasion, and promoted breast cancer cell apoptosis in vitro. Xenograft breast tumor model revealed that knockdown of SEC61G inhibited breast tumor development in vivo. Furthermore, we demonstrated that SEC61G positively regulated glycolysis in breast cancer cells. Mechanistically, we showed that transcription factor E2F1 directly bound to the promoter of SEC61G and regulated its expression in breast cancer cells. SEC61G overexpression antagonized the effect of E2F1 knockdown in regulating breast cancer cell proliferation, invasion, and apoptosis. Finally, we demonstrated that the E2F1/SEC61G axis regulated glycolysis and chemo-sensitivity of Herceptin in breast cancer cells. Taken together, these results of in vitro and in vivo studies demonstrate that SEC61G promotes breast cancer development and metastasis via modulating glycolysis and is transcriptionally regulated by E2F1, which might be utilized as a promising therapeutic target of breast cancer treatment.

Prognostic significance of phosphoglycerate dehydrogenase in breast cancer.

Breast cancer is the most common type of cancer affecting women worldwide. Phosphoglycerate dehydrogenase (PHGDH) is an oxidoreductase in the serine biosynthesis pathway. Although it has been reported to affect growth of various tumors, its role in breast cancer is largely unknown. This study aimed to analyze the expression of PHGDH in breast cancer tissue samples and to determine if PHGDH regulates breast cancer cell proliferation. Tissue microarrays consisting of 305 cases of breast invasive ductal carcinoma were used for immunohistochemical evaluation of PHGDH expression. The role of PHGDH in breast cancer was investigated in vitro by knocking down its expression and determining the effect on cell proliferation and cell cycling, and in ovo by using a chorioallantoic membrane (CAM) assay. Immunohistochemical examination showed that PHGDH is mainly localized in the cytoplasm of breast cancer cells and significantly associated with higher cancer grade, larger tumor size, increased PCNA expression, and lymph node positivity. Analysis of the GOBO dataset of 737 patients demonstrated that increased PHGDH expression was associated with poorer overall survival. Knockdown of PHGDH expression in breast cancer cells in vitro resulted in a decrease in cell proliferation, reduction in cells entering the S phase of the cell cycle, and downregulation of various cell cycle regulatory genes. The volume of breast tumor in an in ovo CAM assay was found to be smaller when PHGDH was silenced. The findings suggest that PHGDH has a regulatory role in breast cancer cell proliferation and may be a potential prognostic marker and therapeutic target in breast cancer.

MiR-301b-3p Regulates Breast Cancer Cell Proliferation, Migration, and Invasion by Targeting NR3C2.

Objectives Breast cancer is the most common malignant tumor among females, and miRNAs have been reported to play an important regulatory role in breast cancer progression. This study aimed to explore the function and underlying molecular mechanism of miR-301b-3p in breast cancer. Methods Differential analysis and survival analysis were performed based on the data accessed from the TCGA-BRCA dataset for identification of the target miRNA. Bioinformatics analysis was conducted to predict the downstream target gene of the miRNA. Real-time quantitative PCR was carried out to detect the expression of miR-301b-3p and nuclear receptor subfamily 3 group C member 2 (NR3C2). Western blot was used to assess the protein expression of NR3C2. Cell counting kit-8 assay was performed to evaluate the proliferation of breast cancer cells. Transwell assay was conducted to determine the migratory and invasive abilities of breast cancer cells. Dual-luciferase reporter assay was employed to verify the targeting relationship between miR-301b-3p and NR3C2. Results miR-301b-3p was elevated in breast cancer cell lines and promoted cell proliferation, migration, and invasion in terms of its biological function in breast cancer. NR3C2 was validated as a direct target of miR-301b-3p via bioinformatics analysis and dual-luciferase reporter assay, and NR3C2 was downregulated in breast cancer cell lines. The rescue experiment indicated that NR3C2 was involved in the mechanism by which miR-301b-3p regulated the malignant phenotype of breast cancer cells. Conclusion The present study revealed for the first time that miR-301b-3p could foster breast cancer cell proliferation, migration, and invasion by targeting NR3C2, unveiling that miR-301b-3p is a novel carcinogen in breast cancer.

MicroRNA miR-92a-3p regulates breast cancer cell proliferation and metastasis via regulating B-cell translocation gene 2 (BTG2)

ABSTRACT MicroRNAs (miRNAs) dysregulation contributes to tumorigenesis, and it is reported that abnormal miR-92a-3p expression participates in multiple cancers’ occurrence and progression. This study focuses on miR-92a-3p’s functions and regulatory mechanism in breast cancer (BC). The current study proved miR-92a-3p expression was enhanced in BC tissues and cells, and its high expression was related to increased TNM stage and larger tumor size of BC patients. Functionally, transfection of miR-92a-3p mimics facilitated BC cell proliferation and metastasis, yet transfection of miR-92a-3p inhibitors functioned oppositely. In addition, BTG2 was verified as a direct miR-92a-3p target in BC cells. This research indicated that miR-92a-3p facilitates BC cell proliferation and metastasis through repressing BTG2 expression.

Upregulation of CCT-3 Induces Breast Cancer Cell Proliferation Through miR-223 Competition and Wnt/β-Catenin Signaling Pathway Activation.

The clinical significance and the function of chaperonin-containing TCP1 complex 3 (CCT-3) in breast cancer remain unknown. In this study, we found that CCT-3 was markedly overexpressed in breast cancer tissues. Statistical analysis revealed a significant correlation of CCT-3 expression with advanced breast cancer clinical stage and poorer survival. Ablation of CCT-3 knocked down the proliferation and the tumorigenicity of breast cancer cells in vitro and in vivo. CCT-3 may regulate breast cancer cell proliferation through a ceRNA network between miR-223 and β-catenin, thus affecting Wnt/β-catenin signaling pathway activation. We also validated that CCT-3 and β-catenin are novel direct targets of tumor suppressor miR-223. Our results suggest that both mRNA and the protein levels of CCT-3 are potential diagnosis biomarkers and therapeutic targets for breast cancer.

Abstract 3976: Tumor-educated osteoblasts regulate breast cancer cell proliferation via extracellular vesicles

Abstract Breast cancer commonly traffics to bone, where breast cancer cells (BCCs) can survive undetected for years before metastatic outgrowth. This period of time can be defined as metastatic latency. In bone, BCCs interact with surrounding stromal cells, including osteoblasts (OBs), to shape the metastatic niche. Our lab discovered that there are at least two distinct subpopulations of OBs in the bone-tumor niche, based on protein marker expression. One group, “educated osteoblasts” (EOs) have engaged in crosstalk with BCCs whereas another group, naive OBs, have not. We have new, novel evidence that EOs regulate BCC proliferation, and may contribute to metastatic latency. The purpose of this study was to determine if extracellular vesicles (EVs) produced by EOs play a role in regulating BCC proliferation. We hypothesized that EVs produced by EOs, would decrease BCC proliferation. We first isolated EO-derived small EVs from cell culture media via ultracentrifugation and characterized the EVs for size, protein marker expression, and density floatation to validate the purity of the EV samples. We next sought to determine the functionality of EO-derived EVs with regards to regulating BCC proliferation. Strikingly, we found that EVs produced by EOs, but not naïve OBs, decreased BCC proliferation in a concentration dependent manner. We have observed this effect in both triple negative and ER-positive BCCs. Furthermore, using an EdU assay, we found that exposure to EO-derived EVs induces BCCs to undergo cell cycle arrest. Interestingly, we found that this cell cycle arrest was maintained as long as the BCCs were cultured in the presence of EO-derived EVs. However, the cell cycle arrest was reversible and BCC proliferation was restored upon removal of EO-derived EVs. We have also seen that exposure to EO-derived EVs leads to increases in BCC expression of the G1 checkpoint proteins, p21 and p27. We next wanted to investigate proliferative signaling pathways that may be deregulated in BCCs following exposure to EO-derived EVs. We found that EO-derived EVs reduce BCC levels of ERK1/2. This is consistent with previous literature indicating that low ERK1/2 levels are implicated in cell cycle arrest and even cancer cell quiescence. Because our data indicate EO-derived EVs induce sustained cell cycle arrest in BCCs, we desired to know if EO-derived EVs protected BCCs from chemotherapy-induced cell death. Excitingly, we have novel preliminary data demonstrating that BCCs exposed to EO-derived EVs and the chemotherapy drug, doxorubicin, have decreased cell death compared to BCCs exposed only to doxorubicin. Altogether, our data suggest that EOs play a crucial role in bone-tumor microenvironment, by regulating BCC proliferation, and may ultimately contribute to metastatic latency. This study underscores the importance of investigating EOs as regulators of breast cancer progression in bone. Supported by NIH R00 CA178177 and Commonwealth of Pennsylvania - Department of Health SAP 4100072566 for KMB. Citation Format: Alison B. Shupp, Karen M. Bussard. Tumor-educated osteoblasts regulate breast cancer cell proliferation via extracellular vesicles [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3976.

MiR-765 Regulates Breast Cancer Cell Proliferation, Apoptosis, and Adriamycin Resistance Through Targeting Epithelial Membrane Protein 3

Epithelial membrane protein 3 (EMP3) regulates cell proliferation, differentiation, and apoptosis. Bioinformatics analysis revealed that miR-765 had complimentary sequence with the 3 -URT of EMP3 mRNA. miR-765 regulates EMP3 and influences breast cancer cell behaviors. However, it is unclear whether this regulation plays a role in affecting drug resistance. Our study assessed miR765's role in EMP3 expression and adriamycin (ADM) resistance of breast cancer. ADM resistant cell line MCF-7/ADM was established and assigned into miR-NC group, miR-765 mimic group, siRNA-NC group, and siRNA-EMP3 group followed by analysis of cell proliferation and apoptosis. miR-765 targets EMP3. The miR-765 level and apoptosis rate in MCF-7/ADM cells were significantly lower, while EMP3 level and cell proliferation were higher than MCF-7 cells. miR-765 mimic or siRNA-EMP3 significantly downregulated EMP3, weakened cell proliferation, increased apoptosis, and decreased ADM resistance. MiR-765 and EMP3 involves in ADM resistance of breast cancer cells. Up-regulation of miR-765 inhibits breast cancer cell proliferation and reduces ADM resistance via regulating EMP3.

ROR2 knockdown suppresses breast cancer growth through PI3K/ATK signaling

The receptor tyrosine kinase like orphan receptor 2 (ROR2) has been implicated in the pathogenesis of a variety of human cancers, including breast cancer. Here, we analyzed the clinical significance of ROR2 in breast cancer (BC) progression, and its function in the regulation of BC cell proliferation and growth. Analysis of ROR2 mRNA levels in 45 BC tissues and adjacent non-tumor tissues revealed that ROR2 expression was significantly increased in BC tissues, and that it correlated with tumor diameter. Kaplan-Meier disease-free survival (DFS) analysis demonstrated that BC patients with higher ROR2 expression had lower DFS. Knockdown of ROR2 suppressed in vitro proliferation of BC cells and promoted apoptosis, while ROR2 overexpression induced BC cell proliferation and suppressed apoptosis. Importantly, ROR2 suppression also reduced the tumor growth in mouse BC xenografts, indicating that ROR2 promotes BC tumorigenesis in vivo. In addition, our data revealed that ROR2 promotes proliferation of BC cells by activating the PI3K/AKT signaling pathway. Together, our results indicate that ROR2 acts as an oncogenic gene in breast cancer, and suggest that the ROR2/PI3K/AKT regulatory network contributes to breast cancer progression.