Tumor Suppressor In Breast Cancer Research Articles

Особливості експресії молекул клітинної адгезії Е-кадгерину в різних молекулярних підтипах інвазивного протокового раку грудної залози

The purpose of the study was to determine the role of E-cadherin in the development and progression of molecular subtypes of invasive ductal breast cancer by assessing the expression of E-cadherin in various clinical and pathological prognostic parameters. Materials and methods. We demonstrated a comprehensive morphological, including immunohistochemical study of 193 cases of invasive ductal breast cancer with the molecular phenotype definition. General histological processing of samples was performed according to standard methods. Immunohistochemical studies for E-cadherin, ER, PR, c-erbB2, Ki-67 were performed according to standardized analytically validated protocols with the necessary controls. The grade of malignancy was determined according to the modified scheme of P. Scarff, H. Bloom and W. Richardson. E-cadherin expression level was quantified in 86 observations using the Qureshi counting system. Comparison of E-cadherin expression in different clinical and pathological parameters was evaluated using Pearson's test χ2. For all types of analysis, differences were considered significant at p <0.05. Results and discussion. E-cadherin expression in ductal breast cancer tissue is significantly lower in cases with lymph node metastases than without metastatic lymph node involvement (χ2 = 4.55, p = 0.031). Low expression of E-cadherin or its absence was associated with pT3 tumors, clinical stage 3, with G2 and G3 malignancies. Loss of E-cadherin expression has an unfavorable prognostic value. E-cadherin expression is associated with the molecular type of invasive breast ductal carcinoma. High E-cadherin expression was common in ER-positive tumors of the luminal A phenotype and was determined in patients of both premenopausal and postmenopausal age, suggesting that ER-positive expression may be involved in the regulation of E-cadherin expression. Low tumor activity of cells of invasive ductal breast cancer of the luminal subtype is accompanied by an increase in the adhesive properties of these cells due to the high level of expression of E-cadherin. E-cadherin is considered an independent marker of triple-negative breast cancer and is characterized by an unfavorable prognosis and short life expectancy. Triple-negative cancer was associated with a significant predominance of patients with low and negative E-cadherin expression (p = 0.011). Conclusion. Thus, E-cadherin is a potent tumor suppressor of breast cancer. According to this role in the progression of breast cancer, it was found that partial or complete loss of E-cadherin expression correlates with an unfavorable prognosis in patients

Withaferin A mediated changes of miRNA expression in breast cancer-derived mammospheres.

Breast cancer is a heterogeneous disease consisting of atypical cell populations that share stem cell-like characteristics associated with therapeutic resistance, disease relapse, and poor clinical outcome. MicroRNAs (miRNA),and small noncoding RNA, are pivotal in the regulation of self-renewal, stemness, and cellular differentiation. Withaferin A (WA), a steroidal lactone, is a major bioactive constituent of Withania somnifera (Solanaceae) known for its anticancer properties. In this study, the effect of WA on modulation of miRNA expression in breast cancer-derived mammosphere was assessed utilizing small RNA sequencing. Treatment with WA inhibited MCF-7 and T47D cells derived mammosphere formation with a significant decrease in CD44, EpCAM, Nanog, OCT4, and SOX2 as markers of self-renewal and stemness. Small RNA sequencing demonstrated a total of 395 differentially expressed miRNAs (DEMs) including 194 upregulated and 201 downregulated miRNAs in WA-treatedMCF-7 mammospheres. Bioinformatics analysis utilizing theKEGG pathway, Gene Ontology enrichment, protein-protein, and miRNA-mRNA interaction network identified altered expression in a few hub genes viz.AKT1, PTEN, MYC, CCND1,VEGFA,NOTCH1, andIGFR1associated with DEMs in WA-treatedmammospheres. Further quantitativeRT-PCR analysis validated the expression of DEMs including miR-549a-5p, miR-1247-5p, miR-124-5p, miR-137-5p, miR-34a-5p, miR-146a-5p, miR-99a-5p, miR-181a-5p, let-7c-5p, and let-7a-5p. In particular, let-7c-5p is designated as a tumor suppressor in breast cancer. An increase in miR-let-7c-5p expression was noted after WA treatment, with a simultaneous decrease in CCND1 and c-MYC at mRNA and protein levels. Taken together, our study demonstrated WA-mediated miRNA expression, in particular, upregulation of miR-let-7c-5p, leads to the inhibition of breast cancer cells derived mammospheres.

Abstract 3463: Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy due to the lack of early diagnosis and limited response to all treatment modalities. Early metastatic dissemination, rich desmoplastic stroma and immunosuppressive infiltrate are some of the features contributing to the poor prognosis of PDAC patients (5-year survival rate of less than 10%). Despite scientific progress in the development of new therapeutic regimes, PDAC patients in low- and middle-income countries have restricted access to high-cost innovative drugs. Recently, attention has been given to the concept of drug repurposing that could meet the need for novel but affordable and widely accessible therapies. Imidazoline receptor 1 Nischarin (NISCH, IR1, IRAS) is an integrin α5-interacting protein that acts as a tumor suppressor in breast cancer through modulation of cancer cell motility and survival. Importantly, NISCH has several clinically approved agonists that are used for the treatment of hypertension and are shown to modulate autophagy and reduce inflammation and fibrosis, which makes NISCH a potentially good therapeutic target for defying PDAC progression. Based on our GSEA analysis of NISCH expression in PDAC patient samples, NISCH was predicted to have an effect on cancer cell focal adhesion assembly and cytoskeletal organization. Therefore, we examined the effects of NISCH agonists on PDAC cells fitness in vitro. A panel of PDAC cell lines was tested in MTT assay to determine the effects of NISCH agonists rilmenidine, clonidine and moxonidine on cell viability. Flow cytometry, western blot and immunofluorescence were used to examine the effects of NISCH agonists on PDAC cell metabolism, migration and cytoskeletal organization. Tg(fli1:EGFP) zebrafish model was used to examine the effects of NISCH agonists on PDAC cell growth and invasion. We found that NISCH was expressed in PDAC cell lines, and that out of the three tested agonists, rilmenidine most potently inhibited cancer cell viability. Treatment with rilmenidine significantly reduced PANC-1, MIA PaCa-2 and BxPC-3 cell attachment to extracellular matrix (collagen type I and fibronectin) and migratory potential in wound healing and transwell assays. Furthermore, treatment with rilmenidine altered the organization of actin cytoskeleton and focal adhesion assembly, potentially through the reduction of intracellular reactive oxygen species (ROS) levels. Importantly, treatment with rilmenidine inhibited invasion of PANC-1 cells in Tg(fli1:EGFP) zebrafish model. With these antimigratory and potentially antimetastatic effects of rilmenidine, our study lays the ground for a more extensive examination of the biological role of NISCH in PDAC progression and implies that its agonists may be good candidates for drug repurposing in this type of cancer. Citation Format: Marijana Pavlovic, Marija Ostojic, Kristina Zivic, Aleksandar Pavic, Tatjana Srdic-Rajic, Jelena Grahovac. Nischarin agonist rilmenidine inhibits pancreatic ductal adenocarcinoma cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3463.

Abstract 856: Coiled-coil domain containing 3 suppresses breast cancer growth by protecting p53 from proteasome-mediated degradation

Abstract Coiled-coil domain containing 3 (CCDC3) was previously shown to regulate liver lipid metabolism as a secretory protein. Our recent studies revealed an intracellular role of CCDC3 as a tumor suppressor in breast cancer (BrC). Bioinformatics datasets analysis showed that CCDC3 is under-expressed in BrCs, while its higher levels are correlated with higher overall survival and lower relapse of cancer patients. CCDC3 is positively correlated with p53 and its target genes. Ectopic CCDC3 markedly suppressed proliferation, colony formation, and xenograft tumor growth by augmenting p53 activity in BrC cells. In contrast, knockdown of endogenous CCDC3 using CRISPR led to reduction of p53 level and activity, consequently promoting the proliferation of BrC cells. Mechanistically, CCDC3 bound to the C-termini of p53 and MDM2, consequently stabilizing p53 in the nucleus and impairing MDM2 recruitment of p53 to the 26S proteosome without inhibiting p53 ubiquitination. Also, knockdown of CCDC3 caused the resistance of BrC cells to 5-FU to some degrees by alleviating p53 response to this drug. p53 induced CCDC3 expression by binding to its promoter specifically in BrC cells. Our results unveil a unique mechanism underlying CCDC3 activation of p53 in a positive feedback fashion to suppress BrC growth and suggest that CCDC3 deficiency could be a factor for drug resistance of BrCs. Citation Format: Hyemin Lee, Caiyue Li, Ji Hoon Jung, Yiwei Zhang, Jieqiong Wang, Chang Liu, Allyson M. Segall, Roger L. Sheffmaker, Shelya X. Zeng, Hua Lu. Coiled-coil domain containing 3 suppresses breast cancer growth by protecting p53 from proteasome-mediated degradation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 856.

The expression and the tumor suppressor role of CLDN6 in colon cancer.

As a member of the tight junction family, CLDN6 is a tumor suppressor in breast cancer, but its role in colon cancer is unknown. In this research, we aimed at revealing the function of CLDN6 in colon cancer. We found that colon cancer tissues lowly expressed CLDN6, and the expression of CLDN6 was negatively correlated with lymph node metastasis. Similarly, CLDN6 was lowly expressed in the colon cancer cell line SW1116, and overexpression of CLDN6 inhibited cell proliferation in vitro and in vivo. Consistently, the migration and invasion abilities of cells were significantly inhibited after CLDN6 overexpression. In addition, we demonstrated that CLDN6 may inhibit the migration and invasion abilities by activating the TYK2/STAT3 pathway. Therefore, our data indicated that CLDN6 acted as a tumor suppressor and had the potential to be regarded as a biomarker for the progression of colon cancer.

RhoBTB3 Regulates Proliferation and Invasion of Breast Cancer Cells via Col1a1.

Breast cancer is the leading cause of cancer-related death in women worldwide, despite medical and technological advancements. The RhoBTB family consists of three isoforms: RhoBTB1, RhoBTB2, and RhoBTB3. RhoBTB1 and RhoBTB2 have been proposed as tumor suppressors in breast cancer. However, the roles of RhoBTB3 proteins are unknown in breast cancer. Bioinformatics analysis, including Oncomine, cBioportal, was used to evaluate the potential functions and prognostic values of RhoBTB3 and Col1a1 in breast cancer. qRT-PCR analysis and immunoblotting assay were performed to investigate relevant expression. Functional experiments including proliferation assay, invasion assay, and flow cytometry assay were conducted to determine the role of RhoBTB3 and Col1a1 in breast cancer cells. RhoBTB3 mRNA levels were significantly up-regulated in breast cancer tissues as compared to in adjacent normal tissues. Moreover, RhoBTB3 expression was found to be associated with Col1a1 expression. Decreasing RhoBTB3 expression may lead to decreases in the proliferative and invasive properties of breast cancer cells. Further, Col1a1 knockdown in breast cancer cells limited the proliferative and invasive ability of cancer cells. Knockdown of RhoBTB3 may exert inhibit the proliferation, migration, and metastasis of breast cancer cells by repressing the expression of Col1a1, providing a novel therapeutic strategy for treating breast cancer.

Retracted] MicroRNA‑133a acts as a tumour suppressor in breast cancer through targeting LASP1

Following the publication of this paper, it was drawn to the Editors' attention by a concerned reader that the western blotting assay data shown in Figs.5B, 5E, 6C and 7A were strikingly similar to data appearing in different form in other articles by different authors. Owing to the fact that the contentious data in the above article had already been published elsewhere, or were already under consideration for publication, prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. The authors were asked for an explanation to account for these concerns, but the Editorial Office did not receive a reply. The Editor apologizes to the readership for any inconvenience caused. [the original article was published in Oncology Reports 39: 473‑482, 2018; DOI: 10.3892/or.2017.6114].

DMT1‐mediated endosome‐mitochondria interactions regulates iron homeostasis and mitochondrial metabolism

Mitochondria‐early endosome (EE) interactions have been shown to facilitate the translocation of iron into mitochondria. Here we show that Divalent Metal Transporter 1 (DMT1) modulates iron exit from endosomes and transport into mitochondria via regulation of EE‐mitochondria interactions. In cancer cells, mitochondria are the ultimate cellular iron sink, where iron can be either stored or used for example to shift cellular metabolism towards glycolysis (Warburg effect), a key adaptive mechanism of cancer cells. Moreover, a gene signature associated with reduced intracellular iron content, including low transferrin receptor (TfR) (anti‐import) and high ferroportin (FPN) (pro‐export) expression levels, has been related to favorable breast cancer prognosis. Similarly, reduced DMT1 expression associates with improved breast cancer patient survival. We evaluated the role of DMT1 in two distinct breast cancer cell lines: estrogen receptor positive T47D and triple‐negative MDAMB231. In both cell lines, we demonstrate colocalization between EE, DMT1 and mitochondria. Interestingly, DMT1 is localized to the surface contact area between endosomes and mitochondria. To demonstrate that DMT1 plays a role in endosome‐mitochondria interactions and Mitochondrial Iron Translocation (MIT), we have generated MDAMB231 as well as T47D CRISPR/Cas9 based DMT1 knockout (KO) stable cell lines. Several lines of evidence show that DMT1 regulates MIT and labile iron pool (LIP) levels via modulation of EE‐mitochondria interactions in MDAMB231 cells. MIT decrease via DMT1 silencing was partially rescued by re‐expression of DMT1 in MDAMB231, but not in T47D cells. MDAMB231 DMT1 KO cells showed increased Ferro‐Orange staining, indicating higher LIP levels, as well as decreased TfR and increased FPN protein levels. Importantly, DMT1 silencing significantly reduced EE‐mitochondria interactions and EE speed in MDAMB231 but not in T47D. Thus, DMT1 regulates MIT and LIP levels via EE‐mitochondria interactions in MDAMB231. These results are in agreement with previous results showing that MDAMB231 display a delay in iron release in comparison to T47D, making them more sensitive to disruptions in MIT. Since mitophagy has been shown to act as a tumor suppressor in breast cancer, we tested whether it could be modulated by DMT1‐mediated MIT. We found that DMT1 silencing increases mitochondrial ferritin, global autophagy marker LC3B and PINK1/Parkin‐dependent mitophagy markers in MDAMB231; levels of all proteins evaluated were rescued to basal levels upon re‐expression of DMT1 in DMT KO cells. Moreover, DMT1 silencing decreases Tom 20 (outer mitochondrial membrane marker) with PMPCB, a known DMT1 interactor that is required for PINK1 turnover. Concurring with the role of DMT1 in mitophagy and iron metabolism, both mitochondrial metabolism and invasive cell migration are significantly impaired by DMT1 silencing and are partially rescued by re‐expression of DMT1. Overall, our results implicate DMT1 in the regulation of EE dynamics and EE‐mitochondria interactions to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cell migration. Thus, we propose DMT1 as a key player associated with aggressive phenotypes in breast cancer

Disruption of ZNF334 promotes triple-negative breast carcinoma malignancy through the SFRP1/ Wnt/β-catenin signaling axis.

Zinc-finger proteins (ZNFs) constitute the largest transcription factor family in the human genome. The family functions in many important biological processes involved in tumorigenesis. In our research, we identified ZNF334 as a novel tumor suppressor of triple-negative breast cancer (TNBC). ZNF334 expression was usually reduced in breast cancerv (BrCa) tissues and TNBC cell lines MDA-MB-231 (MB231) and YCCB1. We observed that promoter hypermethylation of ZNF334 was common in BrCa cell lines and tissues, which was likely responsible for its reduced expression. Ectopic expression of ZNF334 in TNBC cell lines MB231 and YCCB1 could suppress their growth and metastatic capacity both in vitro and in vivo, and as well induce cell cycle arrest at S phase and cell apoptosis. Moreover, re-expression of ZNF334 in TNBC cell lines could rescue Epithelial-Mesenchymal Transition (EMT) process and restrain stemness, due to up-regulation of SFRP1, which is an antagonist of Wnt/β-catenin signaling. In conclusion, we verified that ZNF334 had a suppressive function of TNBC cell lines by targeting the SFRP1/Wnt/β-catenin signaling axis, which might have the potentials to become a new biomarker for diagnosis and treatment of TNBC patients.

Is mir125-a rs12976445 a significant predictor of the development of breast cancer in women?

Cancer is one of the main causes of mortality worldwide and the cancer deaths figure is estimated to reach 11 million by 2030 (Momenimovahed and Salehiniya, 2019). Among the cancers, breast cancer is the most common malignancy in women worldwide affecting more than 1 million women annually (Stuckey, 2011; Ghoncheh, et al., 2016). Breast cancer incidence varies widely, ranging from 27/100,0002 (Central-East Asia and Africa) to 85-94/100,0002 (Australia, North America and Western Europe) indicating that the environmental and genetic factors play significant role in incidence of breast cancer occurrence worldwide (Sancho-Garnier and Colonna, 2019). Considering the development and pathology of breast cancer, it is a highly heterogeneous cancer in its pathological characteristics, some cases showing slow growth with excellent prognosis, while others being aggressive tumors (Tao, et al., 2015). Despite many studies carried out to investigate the etiology of breast cancer, much of the etiology of this disease is unknown, however, many gene polymorphisms of breast cancer have been described as possible neoplasm etiologic factors (Bugano et al., 2008).MicroRNAs (miRNAs) have been reported to have a pivotal role in gene regulation and the control of cancer-related mechanisms such as apoptosis and metastasis. miRNAs are short non-coding RNAs that are involved in post-transcriptional regulation of gene expression. miRNAs are transcribed by RNA polymerase II. The primary transcript is cleaved by the Drosha ribonuclease III enzyme to produce pre-miRNA, which is further cleaved by the cytoplasmic Dicer ribonuclease to generate the mature miRNA. The mature miRNA is incorporated into a RNA-induced silencing complex, which recognizes target mRNAs through imperfect base pairing with the miRNA and most commonly results in translational inhibition or destabilization of the target mRNA. Studies have shown that the miR-125 family acts as a tumor suppressor in breast cancer and inhibits ERBB2, ERBB3 and other target genes at post-transcriptional level. The miR-125 family has three homologues, hsa-miR-125a, hsa-miR-125b-1 and hsa-miR-125b-2 located at (19q.13.41), (11q.23) and (21q.21), respectively. Research shows ERBB2, HUR, ROCK-1, KLF13 and ARID3 genes are controlled by miR-125a. However, increased expression level of the genes is related to decreased expression of miR-125a, in breast cancer. Single nucleotide polymorphisms (SNP) of the miRNA encoding genes are believed to be involved in cancer development (Bahreini et al., 2020). Single nucleotide polymorphisms such as rs143525273, rs12975333, rs10404453 and rs12976445 are effective in miR-125a expression. Due to the increasing incidence of breast cancer and important role of miR-125a rs12976445 in the disease many studies have cited this single nucleotide polymorphism (Tang et al., 2015; Tang et al., 2019; Duna et al., 2007; Inoue et al., 2014; Deng et al., 2017; Bahreini et al., 2021).In this study, blood samples were prepared from two groups: patients with breast cancern (approved by an oncologist) (n=51) and control healthy women (n=50) after obtaining written informed consent form. PCR-RFLP technique was used to determine miR-125a genotype. The data were analyzed using Chi-Square.Our findings indicated that there was significant difference regarding rs12976445 genotype distribution between the control group and patients with breast cancer indicating that rs12976445 might be a predictor of the development of breast cancer in women.

Overexpression of lncRNA SLC16A1-AS1 Suppresses the Growth and Metastasis of Breast Cancer via the miR-552-5p/WIF1 Signaling Pathway

BackgroundBreast cancer (BC) is the most common cancer and the fifth leading cause of cancer mortality with 685,000 deaths worldwide in 2020. Long non-coding RNAs (lncRNAs) are critical in BC carcinogenesis and progression. However, the functional roles and mechanisms of SLC16A1-AS1 in BC are unknown.MethodsThe expression profile of SLC16A1-AS1 in BC patients was investigated using data from The Cancer Genome Atlas (TCGA) database and checked in 80 BC patients, followed by analyzing the prognostic value of SLC16A1-AS1 in the 80 BC patients. The biological functions of SLC16A1-AS1 were further examined in vivo and in vitro after overexpression of SLC16A1-AS1 in BC cells. Possible binding sites between SLC16A1-AS1 and miR-552-5p were predicted by miRDB and those between miR-552-5p and Wnt inhibitory factor-1 (WIF1) were predicted by miRanda, which were confirmed using dual-luciferase reporter assay with mutation. Spearman correlation assay was applied to evaluate the association between genes. Rescue experiments were further applied to investigate the molecular mechanisms involved.ResultsLower SLC16A1-AS1 expression in BC tissues was related to poor prognosis of BC patients. Upregulation of SLC16A1-AS1 suppressed BC cell viability, colony formation, invasion, and migration in vitro and growth in vivo via sponging miR-552-5p to release WIF1.ConclusionSLC16A1-AS1 is a tumor suppressor in BC, and lower SLC16A1-AS1 expression is an indicator of poor prognosis in BC patients. SLC16A1-AS1 inhibits BC carcinogenesis and progression via the SLC16A1-AS1/miR-552-5p/WIF1 pathway. SLC16A1-AS1 represents a novel diagnostic, therapeutic, and prognostic target for BC management.

Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics.

Tumourigenesis and cancer progression require enhanced global protein translation1–3. Such enhanced translation is caused by oncogenic and tumour suppressive events that drive the synthesis and activity of translational machinery4,5. Here we report the surprising observation that leucyl-tRNA synthetase (LARS) becomes repressed during mammary cell transformation and in human breast cancer. Monoallelic genetic deletion of LARS in mouse mammary glands enhanced breast cancer tumour formation and proliferation. LARS repression reduced the abundance of select leucine tRNA isoacceptors, leading to impaired leucine codon-dependent translation of growth suppressive genes including epithelial membrane protein 3 (EMP3) and gamma-glutamyltransferase 5 (GGT5). Our findings uncover a tumour suppressive tRNA synthetase and reveal that dynamic repression of a specific tRNA synthetase—along with its downstream cognate tRNAs—elicits a downstream codon-biased translational gene network response that enhances breast tumour formation and growth.

GNG2 acts as a tumor suppressor in breast cancer through stimulating MRAS signaling

G-protein gamma subunit 2 (GNG2) is involved in several cell signaling pathways, and is essential for cell proliferation and angiogenesis. However, the role of GNG2 in tumorigenesis and development remains unclear. In this study, 1321 differentially expressed genes (DEGs) in breast cancer (BC) tissues were screened using the GEO and TCGA databases. KEGG enrichment analysis showed that most of the enriched genes were part of the PI3K-Akt signaling pathway. We identified GNG2 from the first five DEGs, its expression was markedly reduced in all BC subtype tissues. Cox regression analysis showed that GNG2 was independently associated with overall survival in patients with luminal A and triple-negative breast cancers (TNBC). GNG2 over-expression could significantly block the cell cycle, inhibit proliferation, and promote apoptosis in BC cells in vitro. In animal studies, GNG2 over-expression inhibited the growth of BC cells. Further, we found that GNG2 significantly inhibited the activity of ERK and Akt in an MRAS-dependent manner. Importantly, GNG2 and muscle RAS oncogene homolog (MRAS) were co-localized in the cell membrane, and the fluorescence resonance energy transfer (FRET) experiment revealed that they had direct interaction. In conclusion, the interaction between GNG2 and MRAS likely inhibits Akt and ERK activity, promoting apoptosis and suppressing proliferation in BC cells. Increasing GNG2 expression or disrupting the GNG2–MRAS interaction in vivo could therefore be a potential therapeutic strategy to treat BC.

Abstract P5-09-03: Mieap, a p53-downstream gene, is associated with suppression of breast cancer cell proliferation and better survival

Abstract (Introduction) We previously reported that the mitochondria-eating protein (Mieap), a p53-downstream gene, is involved in mitochondrial quality control (MQC) by repairing or eliminating unhealthy mitochondria. In the breast mass, endogenous cytoplasmic Mieap was detected in 32% of invasive ductal carcinomas (IDCs), 55.6% of ductal carcinomas in situ, and 88.9% of fibroadenomas by immunohistochemistry (IHC). We also demonstrated that the p53/Mieap-regulated MQC pathway was inactivated in 26.1% of IDCs, wherein its impairment resulted in a significantly shorter disease-free survival (DFS) of patients with primary breast cancer. Therefore, in the present study, we aimed to characterize Mieap as a tumor suppressor and determine its association with prognosis using large human breast cancer cohorts. (Materials and methods) We identified 2980 patients from two worldwide large-scale primary breast cancer cohorts: The Cancer Genome Atlas (TCGA; n=1076) and Molecular Taxonomy of Breast Cancer International Consortium (METABRIC; n=1904). Mieap expression and survival data were extracted using the cBioPortal and Broad Institute Firehose websites. Regarding the ER+/HER2- (luminal), HER2+, and ER-/HER2- (triple negative; TN) breast cancer subtypes, 1336, 236, and 267 patients from the METABRIC cohort were found to have the respective subtypes, whereas from the TCGA cohort, 584, 182, and 157 patients had the respective subtypes. We then divided patients into the high and low groups on the basis of median gene expression levels in each cohort and analyzed the association between Mieap expression and clinical outcome. The Kaplan-Meier method was used to compare the survival curves between Mieap-high and Mieap-low groups, and Fisher’s exact test was performed to compare the clinicopathological characteristics for significance. Furthermore, expression of Mieap-associated genes was evaluated by Gene Set Enrichment Analysis (GSEA). (Results) Mieap expression was significantly downregulated in tumors than in normal tissues in TCGA. It was significantly downregulated in p53-mutant tumors in the whole cohort, and in all luminal, HER2+, and TN subtypes of both cohorts, indicating that Mieap expression was dependent on p53 status. Furthermore, BNIP3 and NIX, co-factors of Mieap, were downregulated in tumors than in normal tissues in TCGA. These expressions were positively correlated with Mieap expression. GSEA demonstrated that cell cycle- and proliferation-associated gene sets (Myc Targets, E2F targets, G2M checkpoint, Mitotic spindle etc.) were significantly enriched in Mieap-low tumors than in Mieap-high tumors. Mieap expression was significantly correlated with KI67, a cell proliferation marker. Mitochondria-associated gene sets were enriched in Mieap-high tumors. Mieap was found to be downregulated in advanced cancer stage and/or histological grade consistently in both cohorts. Mieap expression was downregulated in metastatic tumors than in primary tumors. In the METABRIC cohort, we found that the DFS and overall survival of patients with Mieap-high tumors were significantly longer than those of patients with Mieap-low tumors among those with the whole and luminal subtypes. Similarly, in the TCGA cohort, the DFS of patients with Mieap-high tumors was longer among those with the luminal subtype. (Conclusion) In conclusion, we found that the mechanistic role of Mieap was clinically relevant in the two independent cohorts. Specifically, Mieap was found to be p53-dependent, and its downregulation was associated with advanced breast tumors. Moreover, Mieap-high tumors demonstrated favorable breast cancer prognosis, particularly in the luminal subtype, because of Mieap’s control over the cell cycle. Thus, Mieap plays an important role as a tumor suppressor in breast cancer. Citation Format: Manabu Futamura, Yoshihisa Tokumaru, Kazuaki Takabe, Hirofumi Arakawa, Kazuhiro Yoshida. Mieap, a p53-downstream gene, is associated with suppression of breast cancer cell proliferation and better survival [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-09-03.

Hyperactivation of MAPK Induces Tamoxifen Resistance in SPRED2-Deficient ERα-Positive Breast Cancer.

Simple SummaryTamoxifen has been used for more than 40 years to treat breast tumors that are dependent on the hormone estrogen for their growth. However, resistance and recurrence of the tumors during the course of the treatment are common. Understanding the mechanisms that drive tamoxifen resistance and discovering new biomarkers for early detection are keys for designing appropriate personalized therapies. Here, we show that low levels of SPRED2 may be useful as a novel biomarker of tamoxifen resistance. We found that SPRED2 deficiency causes a hyperactivation of the mitogen-activated protein kinases (MAPKs) ERK1/ERK2, which in turn enhances estrogen signaling and diminishes the toxic effects of tamoxifen on breast cancer cells. Treatment with the ERK1/2 inhibitor, ulixertinib, could restore their sensitivity to tamoxifen. Therefore, we propose that patients with estrogen-dependent breast cancer characterized by low expression levels of SPRED2 may be candidates for a combination therapy with tamoxifen and ulixertinib.Breast cancer is the number one cause of cancer-related mortality in women worldwide. Most breast tumors depend on the expression of the estrogen receptor α (ERα) for their growth. For this reason, targeting ERα with antagonists such as tamoxifen is the therapy of choice for most patients. Although initially responsive to tamoxifen, about 40% of the patients will develop resistance and ultimately a recurrence of the disease. Thus, finding new biomarkers and therapeutic approaches to treatment-resistant tumors is of high significance. SPRED2, an inhibitor of the MAPK signal transduction pathway, has been found to be downregulated in various cancers. In the present study, we found that SPRED2 is downregulated in a large proportion of breast-cancer patients. Moreover, the knockdown of SPRED2 significantly increases cell proliferation and leads to tamoxifen resistance of breast-cancer cells that are initially tamoxifen-sensitive. We found that resistance occurs through increased activation of the MAPKs ERK1/ERK2, which enhances the transcriptional activity of ERα. Treatment of SPRED2-deficient breast cancer cells with a combination of the ERK 1/2 inhibitor ulixertinib and 4-hydroxytamoxifen (4-OHT) can inhibit cell growth and proliferation and overcome the induced tamoxifen resistance. Taken together, these results indicate that SPRED2 may also be a tumor suppressor for breast cancer and that it is a key regulator of cellular sensitivity to 4-OHT.

PCDHB15 as a potential tumor suppressor and epigenetic biomarker for breast cancer.

Breast cancer is among the most frequently diagnosed cancer types and the leading cause of cancer-related death in women. The mortality rate of patients with breast cancer is currently increasing, perhaps due to a lack of early screening tools. In the present study, using The Cancer Genome Atlas (TCGA) breast cancer dataset (n=883), it was determined that methylation of the protocadherin β15 (PCDHB15) promoter was higher in breast cancer samples than that in normal tissues. A negative association between promoter methylation and expression of PCDHB15 was observed in the TCGA dataset and breast cancer cell lines. In TCGA cohort, lower PCDHB15 expression was associated with shorter relapse-free survival times. Treatment with the DNA methyltransferase inhibitor restored PCDHB15 expression in a breast cancer cell line; however, overexpression of PCDHB15 was shown to suppress colony formation. PCDHB15 methylation detected in circulating cell-free DNA (cfDNA) isolated from serum samples was higher in patients with breast cancer (40.8%) compared with that in patients with benign tumors (22.4%). PCDHB15 methylation was not correlated with any clinical parameters. Taken together, PCDHB15 is a potential tumor suppressor in cases of breast cancer, which can be epigenetically silenced via promoter methylation. PCDHB15 methylation using cfDNA is a novel minimally invasive epigenetic biomarker for the diagnosis and prognosis of breast cancer.

Regulation of the tumor suppressor PTEN in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer (BCa) in which estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER-2) are not expressed. Although TNBC cases account for approximately 15% of all BCa cases, TNBC patients' prognosis is poor compared with that of other BCa subtypes. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) plays an important role in cell proliferation and migration by negatively regulating the PI3K/Akt pathway. PTEN is one of the most commonly inactivated tumor suppressors in BCa. PTEN inactivity is associated with larger tumor sizes, multiple lymph node metastases, and an aggressive triple-negative phenotype. This review primarily focuses on two key points: (1) PTEN and its function. (2) The regulation of tumor suppressor PTEN in TNBC. We provide a summary of genomic alterations of PTEN in BCa. We further discuss the transcriptional regulation of PTEN and how PTEN is regulated by posttranscription and posttranslational modification, as well as by protein interactions. Finally, we discuss the perspectives of the PTEN protein in TNBC.

SELENOF is a new tumor suppressor in breast cancer.

Epidemiological evidence has indicated an inverse association between selenium status and various types of cancer, including breast cancer. Selenoproteins are the primary mediators of selenium effects in human health. We have previously reported loss of heterozygosity in breast tumor samples of the gene for one of the selenoproteins, SELENOF. The function of SELENOF remains unclear and whether SELENOF levels impact breast cancer risk or outcome is unknown. The mining of breast cancer patient databases revealed that SELENOF mRNA is significantly lower in late-stage tumor samples and lower levels of SELENOF also predict poor patient outcome from breast cancer. Genetically manipulating SELENOF in human breast cancer cells or in the murine mammary gland by overexpression, silencing or knockout impacted cell viability by affecting both proliferation and cell death. Restoring SELENOF can attenuate a number of aggressive cancer phenotypes in breast cancer cells, including clonogenic survival, and enhance the response to drugs or radiation used in breast cancer therapy. Importantly, enhancing SELENOF expression reduced in vivo tumor growth in a murine xenograft model of breast cancer. These data indicate that SELENOF is a new tumor suppressor in breast cancer.

The functions and prognostic value of Kr\xfcppel\u2010like factors in breast cancer

BackgroundKrüppel‐like factors (KLFs) are zinc finger proteins which participate in transcriptional gene regulation. Although increasing evidence indicate that KLFs are involved in carcinogenesis and progression, its clinical significance and biological function in breast cancer are still limited.MethodsWe investigated all the expression of KLFs (KLF1-18) at transcriptional levels by using Oncomine and Gene Expression Profiling Interactive Analysis (GEPIA). The mRNA and protein expression levels of KLFs were also determined by using RT-qPCR and immunohistochemistry, respectively. CBioPortal, GeneMANIA and STRING were used to comprehensive analysis of the molecular characteristics of KLFs. The clinical value of prognostic prediction based on the expression of KLFs was determined by using the KM plotter. The relevant molecular pathways of KLFs were further analyzed by using Gene Set Enrichment Analysis (GSEA) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. Finally, we investigated the effect of KLF2 and KLF15 on biological behavior of breast cancer cells in vitro.ResultsThe expression of KLF2/4/6/8/9/11/15 was significantly down-regulated in breast cancer. The patients with high KLF2, KLF4 or KLF15 expression had a better outcome, while patients with high KLF8 or KLF11 had a poor prognosis. Furthermore, our results showed that KLF2 or KLF15 can be used as a prognostic factor independent on the other KLFs in patients with breast cancer. Overexpression of KLF2 or KLF15 inhibited cell proliferation and migration, and blocked cell cycle at G0/G1 phase, resulting in cell apoptosis.ConclusionsKLF2 and KLF15 function as tumor suppressors in breast cancer and are potential biomarkers for prognostic prediction in patients with breast cancer.

Small non-coding RNA profiling in breast cancer: plasma U6 snRNA, miR-451a and miR-548b-5p as novel diagnostic and prognostic biomarkers.

Breast cancer is a leading cause of cancer-related death in women. Most cases are invasive ductal carcinomas of no special type (NST breast carcinomas). In this prospective, multicentric biomarker discovery study, we analyzed the expression of small non-coding RNAs (mainly microRNAs) in plasma by qPCR and evaluated their association with NST breast cancer. Large-scale expression profiling and subsequent validations have been performed in patient and control groups and compared with clinicopathological data. Small nuclear U6 snRNA, miR-548b-5p and miR-451a have been identified as candidate biomarkers. U6 snRNA was remarkably overexpressed in all the validations, miR-548b-5p levels were generally elevated and miR-451a expression was mostly downregulated in breast cancer groups. Combined U6 snRNA/miR-548b-5p signature demonstrated the best diagnostic performance based on the ROC curve analysis with AUC of 0.813, sensitivity 73.1% and specificity 82.6%. There was a trend towards increased expression of both miR-548b-5p and U6 snRNA in more advanced stages. Further, increased miR-548b-5p levels have been partially associated with higher grades, multifocality, Ki-67 positivity, and luminal B rather than luminal A samples. On the other hand, an association has been observed between high miR-451a expression and progesterone receptor positivity, lower grade, unifocal samples, Ki-67-negativity, luminal A rather than luminal B samples as well as improved progression-free survival and overall survival. Our results indicated that U6 snRNA and miR-548b-5p may have pro-oncogenic functions, while miR-451a may act as tumor suppressor in breast cancer.