Levels In Breast Cancer Tissues Research Articles

Association of ADP‑ribosylation factor family genes with prognosis and immune infiltration of breast cancer.

Breast cancer (BC) is the most common type of cancer found in women. ADP-ribosylation factors (ARFs) are a group of small proteins that bind to GTP and are involved in controlling different cellular functions. The function and evolution of multiple ARFs in BC have remained to be fully elucidated, despite existing studies on this protein family in Homo sapiens and other species. In the present study, a systematic analysis of ARF expression levels in BC tissues compared to normal breast tissues was performed using data from The Cancer Genome Atlas database. The analysis revealed significantly higher expression of ARFs in BC tissues. In addition, the prognostic significance of ARF1 and ARF3-6 expression levels was assessed in patients with BC. Of note, elevated ARF1 expression was associated with reduced rates of distant metastasis-free survival (DMFS), overall survival (OS) and recurrence-free survival (RFS) in affected individuals. Similarly, patients with high expression levels of ARF3 had lower post-progression survival (PPS) rates. In addition, patients with higher ARF4 expression had worse PPS and patients with high ARF5 expression exhibited lower DMFS. Patients with high ARF6 expression had worse DMFS, OS, RFS and predictive power score values. Furthermore, the expression of ARF was found to be strongly linked to the infiltration of various immune cell types, namely dendritic cells, macrophages, neutrophils, CD8+ T cells and B cells. These significant associations offer a solid foundation for the potential utilization of new therapeutic targets and predictive markers for the treatment of BC.

Clinical value of KiSS-1 and MMP-2 expression levels in breast cancer tissue in evaluating prognosis of elderly breast cancer patients after modified radical mastectomy.

We attempted to clarify clinical value of KiSS-1 and MMP-2 levels in breast cancer (BC) tissue in evaluating prognosis of elderly BC patients after modified radical mastectomy (MCM). The data of 192 elderly female BC patients receiving MCM in our hospital from January 2018 to December 2022 were collected. According to prognosis, patients received division into poor prognosis group (n = 43) and good prognosis group (n = 149). The serum CEA level and KiSS-1 and MMP-2 levels in BC tissue received measurement in both groups. The predictive value of KiSS-1 and MMP-2 alone and jointly in adverse prognosis of elderly BC patients after MCM received assessment. Results showed that No statistical significance was exhibited between both groups in general data (P > 0.05). The serum CEA level and MMP-2 expression in BC tissue in poor prognosis group exhibited elevation relative to those in good prognosis group, and KiSS-1 expression in BC tissue in poor prognosis group exhibited depletion relative to that in good prognosis group, indicating statistical significance (P < 0.05). The high-level KiSS-1 might be a protective element for adverse prognosis of elderly BC patients after MCM, and high-level CEA and MMP-2 might be an independent risk element for adverse prognosis of elderly BC patients after MCM (P < 0.05). KiSS-1 and MMP-2 alone and jointly predicted AUC of adverse prognosis in elderly BC patients after MCM were 0.93, 0.802 and 0.958, with certain predictive values; when cutoff values of KiSS-1 and MMP-2 were 6.15 and 2.26, the predictive value was the best. In conclusion, KiSS-1 and MMP-2 levels in BC tissue possess relation to adverse prognosis of MCM. KiSS-1 and MMP-2 levels in elderly BC patients before surgery may be detected in the future to assist in prognosis evaluation of elderly BC patients after MCM.

TFAP2C Activates CST1 Transcription to Facilitate Breast Cancer Progression and Suppress Ferroptosis.

Cystatin SN (CST1) appears to have pro-tumor effects in breast cancer (BC) and is involved in ferroptosis; however, there is no report on the regulation of ferroptosis by CST1 for BC development. The purpose of this study is to investigate the functions and mechanisms operated by CST1 in BC development and ferroptosis. Transcription Factor Activator Protein 2γ (TFAP2C) and CST1 levels in BC tissues and estrogen receptor (ER)+ cells were quantified by RT-qPCR and western blotting. After knocking down TFAP2C and CST1 expression in MCF7 and T47D cells, the proliferation, colony formation ability, apoptosis, and cell cycle were assessed. Ferroptosis was verified by detecting glutathione peroxidase 4 (GPX4) and 4-hydroxy-2-nonenal (4HNE) levels. The kits were used to test Fe2+, reactive oxygen species, malondialdehyde, and glutathione levels, and ultrastructure of mitochondria was observed through transmission electron microscope. Dual-luciferase reporter assay and chromatin immunoprecipitation test were carried out to investigate the interaction of TFAP2C and CST1. A transplanted tumor model was established to explore the function of TFAP2C in tumorigenesis by quantifying TFAP2C, CST1, Ki67, and GPX4 levels through western blotting and immunochemistry after silencing TFAP2C. TFAP2C and CST1 were predominantly expressed in BC cells. Silencing of TFAP2C or CST1 expression suppressed ER+ BC cell proliferation, promoted apoptosis and ferroptosis, and blocked cell cycle transition from G1 phase to S phase. TFAP2C knockdown in transplanted tumors inhibited tumor growth and GPX4 level. Upregulating CST1 nullified the anti-tumor effects of TFAP2C knockdown and TFAP2C promoted CST1 expression through transcription activation. TFAP2C activates CST1 transcription to facilitate BC development and block ferroptosis.

CENPA knockdown restrains cell progression and tumor growth in breast cancer by reducing PLA2R1 promoter methylation and modulating PLA2R1/HHEX axis.

Breast cancer is a lethal malignancy affecting females worldwide. It has been reported that upregulated centromere protein A (CENPA) expression might indicate unfortunate prognosis and can function as a prognostic biomarker in breast cancer. This study aimed to investigate the accurate roles and downstream mechanisms of CENPA in breast cancer progression. CENPA protein levels in breast cancer tissues and cell lines were analyzed by Western blot and immunohistochemistry assays. We used gain/loss-of-function experiments to determine the potential effects of CENPA and phospholipase A2 receptor (PLA2R1) on breast cancer cell proliferation, migration, and apoptosis. Co-IP assay was employed to validate the possible interaction between CENPA and DNA methyltransferase 1 (DNMT1), as well as PLA2R1 and hematopoietically expressed homeobox (HHEX). PLA2R1 promoter methylation was determined using methylation-specific PCR assay. The biological capabilities of CENPA/PLA2R1/HHEX axis in breast cancer cells was determined by rescue experiments. In addition, CENPA-silenced MCF-7 cells were injected into mice, followed by measurement of tumor growth. CENPA level was prominently elevated in breast cancer tissues and cell lines. Interestingly, CENPA knockdown and PLA2R1 overexpression both restrained breast cancer cell proliferation and migration, and enhanced apoptosis. On the contrary, CENPA overexpression displayed the opposite results. Moreover, CENPA reduced PLA2R1 expression through promoting DNMT1-mediated PLA2R1 promoter methylation. PLA2R1 overexpression could effectively abrogate CENPA overexpression-mediated augment of breast cancer cell progression. Furthermore, PLA2R1 interacted with HHEX and promoted HHEX expression. PLA2R1 knockdown increased the rate of breast cancer cell proliferation and migration but restrained apoptosis, which was abrogated by HHEX overexpression. In addition, CENPA silencing suppressed tumor growth in vivo. CENPA knockdown restrained breast cancer cell proliferation and migration and attenuated tumor growth in vivo through reducing PLA2R1 promoter methylation and increasing PLA2R1 and HHEX expression. We may provide a promising prognostic biomarker and novel therapeutic target for breast cancer.

FEATURES OF COL1A1 EXPRESSION IN BREAST CANCER TISSUE OF YOUNG PATIENTS.

In the last decades, the incidence of breast cancer (BCa) in young women has been increasing steadily. The quantitative indicators of expression of collagen, which play important role in stromal microenvironment, and their association with the age and survival rates of BCa patients have not been yet definitively clarified. To investigate the relationship between the COL1A1 gene expression at the mRNA and protein levels in BCa tissue and the clinicopatological features and survival rates of BCa patients of different age groups. The study was conducted on the clinical material of 50 patients with stage I-III BCa. COL1A1 gene expression at the mRNA and protein levels in BCa tissue were studied using the real-time PCR and immunohistochemical methods, as well as the bioinformatic analysis (UALCAN and Kaplan - Meier Plotter databases). The bioinformatic analysis showed that BCa tissue is characterized by 6.0 times (p < 0.05) higher level of COL1A1 mRNA compared to normal breast tissue. The correlation of COL1A1 expression at the mRNA and protein levels with the molecular subtype of neoplasms was demonstrated. According to Kaplan - Meier Plotter database, a low level of expression of COL1A1 protein level in BCa tissue is associated with lower rates of relapse-free survival of patients. The ex vivo study of the clinical material revealed a decrease in COL1A1 protein expression in tumor tissue of young patients with BCa of T3 category (p < 0.0374), low differentiation grade (p < 0.0163) and basal molecular subtype (p < 0.0001). A correlation between the expression of COL1A1 at the mRNA and protein levels and the expression status of estrogen receptors (p < 0.0001) and progesterone receptors (p < 0.0040) was established. The relapse-free 3-year survival rate of young BCa patients is significantly lower in the presence of a low COL1A1 optical density index in the tumor tissue. The identified relationship between COL1A1 expression and such indicators of BCa malignancy as tumor size, differentiation grade, molecular subtype, receptor status, and the recurrencefree survival of patients indicates the prospects of its use to predict the aggressiveness of the BCa course in young patients.

Estimation of gene expression level of CDH1 as a predisposing factor for metastasis in localized and locally advanced and metastatic breast cancer Iraqi patients

Breast cancer (BC) is a prevalent malignancy among women, ranking as the second most commonly diagnosed cancer globally. Notably, a substantial proportion of breast cancer-related fatalities, up to 90 percent, are attributed to the development of distant organ metastases. While Cadherin1 (CDH1) has conventionally been considered a tumor-suppressor gene in cancer research, recent investigations have unequivocally revealed that both CDH1 and its encoded E-cadherin exhibit oncogenic characteristics. The primary focus of this case-control study is to ascertain the involvement of the CDH1 gene in a specific subset of Iraqi female patients. A total of ninety patients sought diagnosis and treatment at the Oncology Teaching Hospital in Medical City and the Oncology Unit at Al-Yarmouk Teaching Hospital in Baghdad. In addition, we included 30 apparently healthy individuals as blood control subjects and another 30 women with benign breast tumors as tissue control subjects for the study. In the initial phase of the study, we conducted a serological analysis using enzyme-linked immunosorbent assay (ELISA) to detect the concentration of E-cadherin in serum samples from two groups of BC patients. The group with locally advanced and metastatic BC exhibited a significantly higher E-cadherin concentration (963.4 ± 89.8 pg/mL) compared to the group with localized BC. In the second part of the research, qRT-PCR was performed to analyze the expression of the CDH1 gene across all sample types. CDH1 was shown to have the greatest fold expression (2.550 ± 0.164) in cases with locally progressed and metastatic BC. Compared to the seemingly healthy control group, the fold expression for localized BC was 1.456 ± 0.055, and for malignant tissue it was 1.886 ± 0.08621. In conclusion, this study provides compelling evidence supporting CDH1 as an oncogene in BC. The significance of CDH1 in BC tumorigenesis underscores its potential for the development of novel detection biomarkers and targeted therapeutic approaches for BC treatment. Notably, CDH1 exhibited elevated expression levels in BC tissues and demonstrated an association with an unfavorable distant metastasis-free survival outcome.

Prognostic value of lncRNA AFAP1-AS1 in breast cancer: a meta-analysis and validated study in Chinese population.

Long non encoding RNA (lncRNA) plays a crucial role in breast cancer. However, the prognostic role of AFAP1-AS1 in breast cancer remains unclear. To investigate the relationship between the expression of long non-coding RNA actin filament-associated protein1 antisense RNA1 (AFAP1-AS1) and prognosis of breast cancer. Meta-analysis was performed to explore the correlation between AFAP1-AS1 and breast cancer. The AFAP1-AS1expression in patients with breast cancer tissue and adjacent normal tissue from 153 patients was determined by qRT-PCR. Bioinformatics and Cox proportional-hazards risk model were used to explore the relationship between expression of AFAP1-AS1 and prognosis. The combined analysis revealed a significant correlation between AFAP1-AS1 expression and both overall survival (hazard ratios, HR = 2.33, 95%Cl: 1.94-2.81, p < 0.001) as well as disease-free survival/progression-free survival (HR = 2.94, 95%CI: 2.35-3.67, p < 0.001). The relation between expression of AFAP1-AS1 and breast cancer was determined in 153 breast cancer and adjacent normal tissues. The findings revealed a significantly higher AFAP1-AS1expression levels in breast cancer tissues compared to adjacent normal tissues (p < 0.001). Additionally, patients exhibiting heightened levels of AFAP1-AS1 expression were correlated with an unfavorable prognosis (HR = 2.35, 95%CI: 1.47-3.74, p < 0.001), which aligns consistently with the findings of the pooled analysis. The subgroup analysis of clinical characteristics revealed a significant association between high expression of AFAP1-AS1 and TNM stage (HR = 1.72, 95%CI: 1.11-2.65, p = 0.015). This study demonstrated that AFAP1-AS1 acts as an oncogene and may serve as a novel prognostic marker for breast cancer, particularly in the Chinese population.

UBE3B promotes breast cancer progression by antagonizing HIF-2α degradation.

Mutations in E3 ubiquitin ligase UBE3B have been linked to Kaufman Oculocerebrofacial Syndrome (KOS). Accumulating evidence indicates that UBE3B may play an important role in cancer. However, the precise role of UBE3B in cancer and the underlying mechanism remain largely uncharted. Here, we reported that UBE3B is an E3 ligase for hypoxia-inducible factor 2α (HIF-2α). Mechanically, UBE3B physically interacts with HIF-2α and promotes its lysine 63 (K63)-linked polyubiquitination, thereby inhibiting the Von Hippel-Lindau (VHL) E3 ligase complex-mediated HIF-2α degradation. UBE3B depletion inhibits breast cancer cell proliferation, colony formation, migration, and invasion in vitro and suppresses breast tumor growth and lung metastasis in vivo. We further identified K394, K497, and K503 of HIF-2α as key ubiquitination sites for UBE3B. K394/497/503R mutation of HIF-2α dramatically abolishes UBE3B-mediated breast cancer growth and lung metastasis. Intriguingly, the protein levels of UBE3B are upregulated and positively correlated with HIF-2α protein levels in breast cancer tissues. These findings uncover a critical mechanism underlying the role of UBE3B in HIF-2α regulation and breast cancer progression.

Calycosin inhibits triple-negative breast cancer progression through down-regulation of the novel estrogen receptor-α splice variant ER-α30-mediated PI3K/AKT signaling pathway

BackgroundTriple-negative breast cancer (TNBC) is a heterogeneous carcinoma characterized by the most aggressive phenotype among all breast cancer subtypes. However, therapeutic options for TNBC patients have limited clinical efficacy due to lack of specific target and efficient targeted therapeutics. AimTo investigate the biological characteristics of a novel estrogen receptor (ER)-α splice variant ER-α30 in breast cancer cells, and its possible role in the anticancer effects of calycosin, a typical phytoestrogen derived from the herbal plant Astragalus membranaceus, against TNBC. This may also provide a better understanding of the inhibitory activity of calycosin on TNBC progression. MethodsBreast cancer tissues and para-cancer tissues were collected and analyzed for the expression levels of ER-α30 using immunohistochemistry (IHC), and its expression in two TNBC cell lines (MDA-MB-231 and BT-549) was detected by western blot and qRT-PCR assays. Then the alteration of cell viability, apoptosis, migration, invasion and epithelial-mesenchymal transition (EMT) in response to overexpression or knockdown of ER-α30 was separately determined by CCK-8, Hoechst 33258, wound healing, transwell and western blot assays in two TNBC cell lines. Next, the anticancer effects of calycosin on MDA-MB-231 cells were evaluated through CCK-8, colony formation, flow cytometry, Hoechst 33258 and western blot assays, along with the role of ER-α30 in these effects and the possible downstream targets of ER-α30. In addition, the in vivo experiments were carried out using MDA-MB-231 xenograft model intraperitoneally treated with calycosin. The volume and weight of xenograft tumor were measured to evaluate the in vivo anticancer activities of calycosin, while the corresponding changes of ER-α30 expression in tumor tissues were detected by IHC. ResultsIt was demonstrated that the novel ER-α splice variant ER-α30 was primarily distributed in the nucleus of TNBC cells. Compared with normal breast tissues, ER-α30 expression was found in significantly higher levels in breast cancer tissues of ER- and progesterone receptor (PR)-negative subtype, so did in TNBC cell lines (MDA-MB-231 and BT-549) when compared to normal breast cell line MCF10A. Moreover, ER-α30 overexpression strikingly enhanced cell viability, migration, invasion and EMT progression and reduced apoptosis in TNBC cells, whereas shRNA-mediated knockdown of ER-α30 revealed the opposite results. Notably, calycosin suppressed the expression of ER-α30 in a dose-dependent manner, accompanied with the inhibition of TNBC growth and metastasis. A similar finding was observed for the xenografts generated from MDA-MB-231 cells. The treatment with calycosin suppressed the tumor growth and decreased ER-α30 expression in tumor tissues. Furthermore, this inhibition by calycosin was more pronounced in ER-α30 knockdown cells. Meanwhile, we found a positive relationship between ER-α30 and the activity of PI3K and AKT, which could also be inactivated by calycosin treatment. ConclusionFor the first time, it is demonstrated that the novel estrogen receptor-α splice variant ER-α30 could function as pro-tumorigenic factor in the context of TNBC by participating in cell proliferation, apoptosis, invasion and metastasis, thus it may serve as a potential therapeutic target for TNBC therapy. Calycosin could reduce the activation of ER-α30-mediated PI3K/AKT pathway, thereby inhibited TNBC development and progression, suggesting that calycosin may be a potential therapeutic option for TNBC.

Clinical Significance of ABCG2/BCRP Quantified by Fluorescent Nanoparticles in Breast Cancer Patients Undergoing Neoadjuvant Chemotherapy

Breast cancer resistance protein (BCRP), also known as ATP-binding cassette transporter G2 (ABCG2), is associated with chemotherapy resistance. BCRP is also implicated in breast cancer stem cells, and is reported as a poor prognostic factor. However, the relationship of BCRP levels in breast cancer tissues with chemotherapy resistance and prognosis has not been clarified. We aimed to evaluate the correlation between BCRP expression and prognosis in breast cancer using immunohistochemistry with fluorescent phosphor-integrated dots (IHC-PIDs). A total of 37 breast cancer patients with residual cancer in the primary tumor and axillary lymph nodes were evaluated. BCRP levels in breast cancer tissue and metastatic lymph nodes were quantitatively detected after neoadjuvant chemotherapy (NAC). Among these 37 patients, 24 had corresponding core needle biopsies obtained before NAC. Biomarker assay with IHC-PIDs showed high accuracy for the quantitative assessment of BCRP with low expression. High BCRP expression in the primary tumor and metastatic lymph nodes after preoperative chemotherapy was associated with worse overall survival. In conclusion, high BCRP levels may be associated with poor prognosis in patients with breast cancer, having residual tumors within the primary tumor and lymph nodes after preoperative chemotherapy. These findings provide a basis for further appropriate adjuvant therapy in these patients.

Functional variant rs10175368 which affects the expression of CYP1B1 plays a protective role against breast cancer in a Chinese Han population.

Cytochrome P450 1B1 ( CYP1B1 ) genetic variants are relevant in the pathogenesis of breast cancer. Exploring the relationships between CYP1B1 functional variants and breast cancer could improve our understanding of breast cancer molecular pathophysiology. This is a two-stage hospital-based case-control study of a Chinese Han population. Genotyping was performed to identify candidate gene variants. 3DSNP, ANNOVAR, and RegulomeDB were used to determine functional single nucleotide polymorphisms (SNPs). The relationship between candidate variants and breast cancer risk was evaluated through unconditional logistic regression analysis. The PancanQTL platform was used to perform cis and trans expression quantitative trait loci (eQTL) analysis of positive SNPs. The GSCA platform was then used to compare the gene expression levels of potential target genes between breast cancer tissue and normal tissue adjacent to the cancer. rs10175368-T acted as a protective factor against breast cancer based on an additive model [odds ratio (OR) = 0.722, 95% confidence interval (CI) = 0.613-0.850; P < 0.001], and was identified as a protective factor in the postmenopausal population (OR = 0.601; 95% CI, 0.474-0.764; P < 0.001). eQTL analysis and analysis of differential expression in carcinoma and paracancerous tissues revealed that the expression level of CYP1B1 - AS1 was associated with rs10175368 and that CYP1B1-AS1 had significantly higher expression levels in breast cancer tissues than in paracancerous tissues. We show, for the first time in a Chinese Han population, that the functional variant rs10175368 plays a protective role against breast cancer, especially in the postmenopausal population.

Abstract P3-05-15: Clinical significance of breast cancer resistance protein (BCRP) quantified by fluorescence nanoparticle in breast cancer patients undergoing neoadjuvant chemotherapy

Abstract Breast cancer resistance protein (BCRP), also known as ATP-binding cassette transporter G2 (ABCG2), is associated with chemotherapy resistance. BCRP is also implicated in breast cancer stem cells and has been reported as a poor prognostic factor. However, the relationship of BCRP levels in breast cancer tissues with chemotherapy resistance and prognosis has not been clarified to date. Our laboratory recently developed nanoparticles suitable for the quantitative immunohistochemical (IHC) method called phosphor-integrated dots (PIDs). Using PID-based IHC (IHC-PIDs), many biomarker proteins can be visualized and quantitatively analyzed at the single-particle level in paraffine-embedded, formalin-fixed tumor samples. We aimed to evaluate the correlation between quantitatively analyzed BCRP expression and prognosis in breast cancer tissue samples using immunohistochemistry with IHC-PIDs. Thirty-seven Japanese patients diagnosed with invasive ductal carcinoma of the breast who underwent mastectomy and lymph node dissection at Tohoku University Hospital (Sendai, Japan) between 2004 and 2010 were included. All patients had undergone NAC and were pathologically diagnosed with non-pathological complete response or progressive disease and lymph node metastasis postoperatively. The patients were followed up within a median of 10.1 years (range, 1.1–18.2 years). Among the thirty-seven patients, twenty-eight patients had core needle biopsy specimens taken before NAC. BCRP levels in breast cancer tissue and metastatic lymph nodes were quantitatively detected after preoperative chemotherapy and core needle biopsy. Biomarker assay with IHC-PIDs showed high accuracy for quantitative assessment of BCRP with low expression. The optimal PT+LN BCRP cut-off score for predicting survival was 27.4, with a sensitivity of 60.6% and a specificity of 77.3%. BCRP expression in PT+LN samples was dichotomized into high and low groups at this cutoff value, and OS was compared between these group. The results showed that OS was significantly worse in the high BCRP-expression group (log-rank p=0.0089). In conclusion, high BCRP levels are associated with poor prognosis in patients with breast cancer with residual tumors within the primary tumor and lymph nodes after preoperative chemotherapy. These findings provide a basis for further appropriate adjuvant therapy in these patients. Citation Format: Hiroshi Tada, Kohsuke Gonda, Narufumi Kitamura, Minoru Miyashita, Narumi Harada-Shoji, Yohei Hamanaka, Akiko Ebata, Takanori Ishida. Clinical significance of breast cancer resistance protein (BCRP) quantified by fluorescence nanoparticle in breast cancer patients undergoing neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-05-15.

Abstract P2-26-20: Marvel D3 and its associated junctional proteins in breast cancer

Abstract Marvel D3 and its associated junctional proteins in breast cancer Wenxiao Ji, Wen G. Jiang, Tracey A. Martin CCMRC, Cardiff University School of Medicine, Cardiff, Wales, UK Introduction. Marvel (Membrane Associated Domain Containing) proteins are a small family of proteins that are concentrated at the tight junction (TJ) region of epithelial and endothelial cells. They are important players in the formation and regulation of TJs. The Marvel protein family has three members, Marvel-D1, Marvel-D2 and Marvel-D3, all known to be TJ components. Marvel-D3 is more prominent in TJ regulation, by interacting widely with other proteins that co-localised at TJs. In the present study, we have examined the expression of Marvel-D3 and its splicing variants, their relationship with other known interactive TJ partners in breast cancer and in disease progression and clinical outcome. Methods. Marvel-D3 and its variants were quantified in a breast cancer cohort that contained both normal and tumour tissues. The expression was analysed together with the known Marvel-D3 interactive molecules available in our database of the same cohort. The profile of the expression of the molecules was also integrated to search for a pattern of the expression that may have clinical significance including the survival of the patients. Results. Marvel-D3 had significantly high levels in breast cancer tissues than normal tissues (p=0.011). The difference between normal and tumour tissues for two Marvel-D3 variants were not statistically significant. Of all the known Marvel-D3 interactive proteins that were available for analyses, we identified thirteen with viability in integrated analyses; they include the JAM family members, Marvel-D2, occludin, the zonula occluden family members, small number of the claudin family members and cingulin, all being important TJ components. The integrated expression of these Marvel-D3 partners presented a highly significant predictive power for the overall survival of the patients (RUC=0.821, p&amp;lt; 0.0001). Stratifying the patients based on the profile has a significant value in evaluating survival (survival rate 95.7% with favourable expression versus 57.1% unfavourable, during the followup period). This predictive power is independent of other clinical and hormonal receptor status (p&amp;lt; 0.001, HR=1.226 (95% CI 1.094-1.373)). We have also noted that this predictive value is applicable to subtypes of the breast cancer and hormone receptor status with similar predictive power. Conclusion. Expression of Marvel-D3, a TJ component, together with its interactive TJ partners, including both transmembrane and intracellular subcoat proteins, form an important clinical indicator for clinical progression of breast cancer. Citation Format: WENXIAO JI, Wen G. Jiang, Tracey A. Martin. Marvel D3 and its associated junctional proteins in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-26-20.

Long Non-coding RNA LINC00473 Promotes Breast Cancer Progression via miR-424-5p/CCNE1 Pathway.

There has been a large increase in the incidence of breast cancer (BC) among women. LINC00473 is a cancer-related lncRNA, participating in the progression of many cancers, but its role in the progression of BC awaits more elaboration. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to quantify LINC00473, miR-424-5p, and cyclin E1 (CCNE1) mRNA expression levels in BC tissues and cells. Cell counting kit-8 (CCK-8) assay was employed to detect the cell viability; the cell migration and invasion abilities were evaluated by the Transwell assay. Western blot and immunohistochemistry (IHC) were adopted to study CCNE1 protein expression; dual-luciferase reporter assay was performed to clarify the targeting relationships among LINC00473, miR-424-5p, and CCNE1. LINC00473 expression was elevated in BC tissues and cell lines, which was associated with lymph node metastasis and higher clinical stage of the patients with BC. LINC00473 proved to be a molecular sponge for miR-424-5p; LINC00473 knockdown impeded the growth, migration, invasion, and epithelial-mesenchymal transition of BC cells, while these effects were abolished by miR-424-5p inhibitors; miR-424-5p targeted CCNE1 to restrain its expression. LINC00473 positively regulated CCNE1 expression, and CCNE1 restoration counteracted the effects induced by LINC00473 knockdown in BC cells. LINC00473 facilitates the progression of BC through miR-424-5p/CCNE1 axis.

PATTERN OF MMP2 AND MMP9 EXPRESSION DEPENDS ON BREAST CANCER PATIENTS' AGE.

Despite the large number of studies devoted to the study of the features of tumor microenvironment in breast cancer (BCa), presently there is no consensus on the features of MMP-2 and MMP-9 expression in the tumor tissue of BCa patients depending on the age. The aim of the study was to investigate the relationship between MMP-2 and -9 expression at the protein and mRNA levels in BCa tissues and the clinical and pathological features of BCapatientsin different age groups. The expression level of MMP-2 and -9in the BCa tissue of patients of two age groups (< 45 years and > 45 years) was studied using the bioinformatics method (UALCAN database), immunohistochemical method, and real-time PCR. It was established that a characteristic feature of BCa in young patients is the low level of MMP2 mRNA against the background of increased expression of this gelatinase at the protein level, as well as decreased expression of MMP9 at both the mRNA and protein levels. When analyzing the correlation of the gelatinase expression indices in BCa tissue of young patients, depending on the clinical and pathological features, a significantly lower level of MMP-2 expression was recorded in BCa cases of stage II compared to the indices of stage I cases. High expression of MMP-2 and -9 was recorded in BCa tissue in node-positive cases and the basal molecular BCa subtype. The identified relationship between the expression of the studied gelatinases and such indices of BCa malignancy as its stage, positive regional lymph node status, and the molecular BCa subtype in young patients indicates the need for further research of the features of the tumor microenvironment to predict the cancer aggressiveness.

Doxorubicin resistance in breast cancer xenografts and cell lines can be counterweighted by microRNA-140-3p, through PD-L1 suppression.

Doxorubicin, a first-line chemotherapeutic drug for breast cancer, kills cancer cells by inducing DNA-crosslinking damage. Dysregulated micro-RNA (miRNA) is associated with the drug resistance of tumors. However, little is known about the effect of miRNA-140-3p on DOX resistance of breast cancer. The miRNA microarray was used to sequence the transcripts of DOX-chemoresistant breast cancer tissues and DOX-chemosensitive tissues. Then, the breast cancer tissue chip in the GEO database was also analyzed to screen the target gene. Flow cytometry, in situ hybridisation (ISH), immunohistochemistry (IHC), Western blot, cell proliferation assay, real-time PCR analyses (qRT-PCR), and pull-down assay were used to explore the effects of miRNA-140-3p and programmed death ligand-1 (PD-L1) on the chemoresistance of DOX-resistant breast cancer cells treated with DOX. In vivo, the DOX-resistant breast cancer cell lines treated with miRNA-140-3p overexpression were injected subcutaneously into mice to construct breast cancer subcutaneous xenograft tumor models. Based on miRNA microarray, GEO database, and bioinformatics analysis, it was found that miRNA-140-3p and PD-L1 are the core molecules in the DOX resistance regulatory network in breast cancer, and lower miRNA-140-3p and higher PD-L1 expression levels were observed in DOX-resistant breast cancer tissues and cells. IHC results showed that compared with breast cancer tissues with high miRNA-140-3p expression, PD-L1 protein expression levels in breast cancer tissues with low miRNA-140-3p were significantly higher (P<0.01). Moreover, compared with DOX-sensitive tissues, the levels of PD-L1 protein expression in DOX-resistant tissues were significantly higher (P<0.01). In in vitro and in vivo experiments, the introduction of miRNA-140-3p decreased PD-L1 expression. Mechanically, we found that the MCF-7/DOX and HS598T/DOX cells pretreated with miRNA-140-3p inhibitor or exosomes containing PD-L1 have higher stemness and lower apoptosis rate, which can be abrogated by co-treating cells with anti-PD-L1 antibody or miRNA-140-3p mimic. MiRNA-140-3p can suppress PD-L1 expression in breast cancer cell-derived exosomes, thereby attenuating the chemoresistance induced by DOX in breast cancer.

ODP562 ZnT5 Involvement in EMT in Breast Cancer

Abstract The zinc levels in breast cancer tissue are reportedly higher than in normal tissue. In addition, the expression levels of zinc transporters, including ZnT5 and ZnT6, in breast cancer tissue are higher than in normal breast tissue. Moreover, ZnT5 and ZnT6 contribute to the formation of heterodimers and are involved in different biological functions. In vitro studies using chicken DT40 cells showed that ZnT5 and ZnT6 heterodimers are required to activate several proteins that play important roles in tumorigenesis, infiltration, and metastasis. However, the functions of ZnT5 and ZnT6 heterodimers in breast cancer remain unknown. Therefore, in this study, we first immunolocalized ZnT5 and ZnT6 in 113 pathological specimens of breast cancer. We then analyzed the interaction between ZnT5 and ZnT6 in breast cancer tissue using proximity ligation assay (PLA). Further, we evaluated cell migration and the expression of epithelial mesenchymal transition (EMT) markers as well as vimentin and E-cadherin in ZnT5 knockdown MCF-7 cells using siRNA. Next, we utilized human phosphorylation multipathway profiling array to explore the underlying mechanism of ZnT5 knockdown-induced cell migration. Immunohistochemical analysis revealed that the number of ZnT5-positive breast cancer cells was significantly higher in patients with a low pathologic N factor, and the number of ZnT6-positive breast cancer cells was significantly higher in patients with a low histological grade. The number of ZnT5 and ZnT6 double-positive breast cancer cells was significantly higher in patients with low Ki-67 expression than those with ZnT5-positive and ZnT6-negative breast cancer cells. In addition, ZnT5 and ZnT6 heterodimers were detected using PLA in breast cancer tissues with high expression of both ZnT5 and ZnT6. Treatment with 100 µM ZnCl 2 inhibited migration of MCF-7 cells, but ZnT5 knockdown promoted cell migration. ZnT5 knockdown induced higher levels of vimentin and lower levels of E-cadherin and activated SMAD1 in the presence of 100 µM ZnCl 2 . These results indicate that the involvement of ZnT5 in the inhibition of EMT through SMAD1 inactivation. These results also indicate that the role of ZnT5 and ZnT6 heterodimers differs from that of the ZnT5 homodimer in cell proliferation. However, further studies are required to clarify the role of zinc transporters and zinc signaling in breast cancer. Presentation: No date and time listed

Leptin and leptin receptor expression in breast carcinomas and their relationship with clinicopathological features

ContextBreast cancer is the most common cancer and the most common cause of death from cancer among women. Leptin is thought to play role in cell proliferation. ObjectiveIn this study, we evaluated the expression of leptin and leptin receptor in breast cancer and normal breast tissue, analyzed the relationship between expression of leptin, leptin receptor and clinicopathological features in breast cancers. Materials and MethodsThe expression of leptin and leptin receptor were investigated by immunohistochemistry in 205 breast cancer and 30 normal breast tissue. ResultsBreast cancers showed higher expression of leptin (p = 0,003) and leptin receptor (p < 0,001) than normal breast tissues. The expression of leptin showed significant correlation with leptin receptor expression (r = 0,644; p < 0,001). Higher rates of ER and PR positivity are found in leptin positive tumors(p < 0,001). Expression levels of leptin were positively corelated with ER (r = 0,336; p < 0,001) and PR (r = 0,359; p < 0,001) expression. HER2 positivity rates were lower in leptin positive tumors (p = 0,020). Negative correlation between leptin expression levels and histological grade was found (r = -0,168; p = 0,016). Higher rates of leptin receptor positivity were detected in invasive ductal carcinoma NSTs than invasive lobular carcinomas. ConclusionElevated expressions of leptin and leptin receptor levels in breast cancer tissues suggest that leptin may have a promoting effect on the carcinogenesis of breast cancer, possibly in an autocrine manner. Inhibition of leptin may be effective for the treatment of breast cancer.

CUX2/KDM5B/SOX17 Axis Affects the Occurrence and Development of Breast Cancer.

Abnormal expression of CUT-like homeobox 2 gene (CUX2) has been highlighted as potential clinical biomarkers in human cancers. Notably, the function of CUX2 has been less elucidated in breast cancer (BC). We focused on the role of the CUX2 in tumorigenesis and progression of BC with the involvement of the lysine demethylase 5B (KDM5B)/sex determining region Y-box 17 (SOX17) axis. CUX2, KDM5B, and SOX17 expression levels in BC tissues and cells were tested by reverse transcription quantitative PCR and Western blotting. Later, the effects of CUX2, KDM5B, and SOX17 on the malignant behaviors of MDA-MB-231 and MCF-7 cells were analyzed by CCK-8, colony formation, and Transwell assays in vitro. The interactions of CUX2, KDM5B, and SOX17 were validated by online website prediction, ChIP assay, and dual luciferase reporter gene assay. The subcutaneous tumorigenesis in nude mice was conducted to observe the roles of CUX2, KDM5B, and SOX17 in BC tumor growth in vivo. CUX2 and KDM5B were highly expressed while SOX17 had low expression in BC. Inhibition of CUX2 suppressed BC cell malignant phenotypes. CUX2 promoted KDM5B expression through transcriptional activation, enabling its high expression in BC. KDM5B inhibited SOX17 expression through histone demethylation. Overexpression of KDM5B or downregulation of SOX17 reversed the inhibitory effect of CUX2 downregulation on the malignant behaviors of BC cells. Inhibition of CUX2 impeded BC cell growth in vivo through the KDM5B/SOX17 axis. This study highlights that suppression of CUX2 inhibits KDM5B to repress tumorigenesis and progression of BC through overexpressing SOX17.

Inhibition of cell invasion and migration by targeting matrix metalloproteinase-9 expression via sirtuin 6 silencing in human breast cancer cells

Sirtuin 6 (SIRT6) regulation is involved in carcinogenesis. However, its role in breast cancer (BC) metastasis remains unclear. We investigated the effects of SIRT6 on protein kinase C activator- and cytokine-mediated cancer cell invasion and migration in MCF-7 and MDA-MB-231 cells and the association between SIRT6 and matrix metalloproteinase-9 (MMP-9) expression. To assess MMP-9 and SIRT6 expression in patients, protein levels in BC tissues were analyzed. MCF-7 and MDA-MB-231 cell viability was analyzed using MTT assays. SIRT6 was silenced in both cell lines and protein secretion, expression, and mRNA levels were analyzed. Transcription factor DNA activity was investigated using luciferase assays. Matrigel invasion assays were used to assess the effects of SIRT6 in both cell lines. SIRT6 and MMP-9 expression in cancer tissues was significantly higher than in paired normal breast tissues. 12-O-tetradecanoylphorbol-13-acetate (TPA) or tumor necrosis factor-α (TNF-α) increased MMP-9 expression and cell invasion and migration, but SIRT6 knockdown abolished these effects. SIRT6 overexpression additively increased TPA- and TNF-α-induced MMP-9 expression. SIRT6 knockdown suppressed the mitogen-activated protein kinase (MAPK) signaling pathway and thus TPA- and TNF-α-induced MMP-9 expression. SIRT6 silencing suppressed TPA- and TNF-α-induced nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) expressions in both cell lines, and treatment with MAPK, NF-κB, and AP-1 inhibitors reduced MMP-9 expression. The anti-invasive effects of SIRT6 in BC cells might be mediated by suppression of MAPK phosphorylation and reduction in NF-κB and AP-1 DNA activities, leading to MMP-9 downregulation, suggesting that SIRT6 modulation has the potential to target BC metastasis.