Paired Breast Cancer Research Articles

Evaluation of MYC Oncogene Expression in Human Breast Cancer and Its Relationship with ACSL4 and Lipin-1 Expression.

Disturbances in lipid metabolism are one of the hallmarks of cancer cells. Fatty acid synthesis and oxidation play a crucial role in the proliferation, growth, and survival of cancer cells. Several enzymes are involved in lipid metabolism. MYC is an oncogene and plays various regulatory roles in lipid metabolism. This study aimed to evaluate MYC expression and its association with the expressions of Lipin-1, ACSL4 (enzymes involved in lipid metabolism), in pairs of breast cancer (BC) and adjacent normal tissues to further understand the MYC influence on metabolic regulation. Fifty-five pairs of samples of BC and noncancerous adjacent tissues were utilized in the present study to analyze MYC, Lipin-1, and ACSL4 by quantitative real-time polymerase chain reaction. Further, the expression of Lipin-1 and ACSL4 proteins and a number of other clinicopathologically relevant variables were studied employing immunohistochemistry staining. MYC expression was substantially higher in BC tissues than in adjacent normal tissues, according to our findings. This upregulation was positively correlated with tumor size and stage. Although MYC expression was not correlated with that of ACSL4 and Lipin-1 expression, this may result from the complex metabolic changes that occur when cells become malignant. Although further research is required to assess MYC's impact on tumor metabolic regulation, the correlations seen here between MYC, the pathological stage, and tumor size may indicate its prognostic significance in BC. Hence, it may be considered as a potential therapeutic target for further studies.

Computational Analyses Reveal Deregulated Clock Genes Associated with Breast Cancer Development in Night Shift Workers.

Breast cancer (BC) is the leading cause of cancer death among women worldwide. Women employed in shift jobs face heightened BC risk due to prolonged exposure to night shift work (NSW), classified as potentially carcinogenic by the International Agency for Research on Cancer (IARC). This risk is linked to disruptions in circadian rhythms governed by clock genes at the cellular level. However, the molecular mechanisms are unclear. This study aimed to assess clock genes as potential BC biomarkers among women exposed to long-term NSW. Clock gene expression was analysed in paired BC and normal breast tissues within Nurses' Health Studies I and II GEO datasets. Validation was performed on additional gene expression datasets from healthy night shift workers and women with varying BC susceptibility, as well as single-cell sequencing datasets. Post-transcriptional regulators of clock genes were identified through miRNA analyses. Significant alterations in clock gene expression in BC compared to normal tissues were found. BHLHE40, CIART, CLOCK, PDPK1, and TIMELESS were over-expressed, while HLF, NFIL3, NPAS3, PER1, PER3, SIM1, and TEF were under-expressed. The downregulation of PER1 and TEF and upregulation of CLOCK correlated with increased BC risk in healthy women. Also, twenty-six miRNAs, including miR-10a, miR-21, miR-107, and miR-34, were identified as potential post-transcriptional regulators influenced by NSW. In conclusion, a panel of clock genes and circadian miRNAs are suggested as BC susceptibility biomarkers among night shift workers, supporting implications for risk stratification and early detection strategies.

Somatic and germline aberrations in homologous recombination repair genes among Chinese high-risk breast cancer patients by multi-gene next-generation sequencing.

Recently, genes involved in homologous recombination repair (HRR) pathway have been extensively studied. However, the landscapes of HRR gene mutations remain poorly defined in Chinese high-risk breast cancer (BC) patients. Our study aims to identify the status of germline and somatic HRR gene mutations and their association with clinicopathological features in these patients. A total of 100 high-risk BC patients from our institution who underwent paired peripheral blood germline and BC tissues somatic 26 genes next-generation sequencing (NGS) from January 2018 to July 2023 were enrolled for retrospective analysis. Out of 100 high-risk BC patients, 55 (55%) had at least one germline or somatic mutation in HRR genes. Among them, 22% carried germline pathogenic variants (19 BRCA1/2 and 3 non-BRCA genes), 9% harbored somatic pathogenic mutations (3 BRCA1/2 and 6 non-BRCA genes). Among high-risk factors, family history and early onset BC showed a correlation with HRR gene mutations (p < 0.05). BRCA1 germline and HRR gene somatic mutations showed a correlation with TNBC, but BRCA2 germline mutations were associated with Luminal B/HER2-negative BC (p < 0.05). Patients with HRR gene somatic pathogenic variant more likely had a lympho-vascular invasion and distant metastasis (p < 0.05). The prevalence of HRR gene germline and somatic mutations were higher in Chinese BC patients with high risk factors. We strongly recommend that these high-risk BC patients receive comprehensive gene mutation testing, especially HRR genes, which are not only related to genetic consultation for BC patients and provide a theoretical basis for necessary prevention and individualized treatment.

Abstract PO4-08-09: Concordance analysis of paired breast cancer core needle biopsies and surgical excision samples using the Oncotype DX Breast Recurrence Score® assay

Abstract INTRODUCTION: The Oncotype DX Breast Recurrence Score® (RS) assay is validated to predict risk of distant recurrence at 10 years and the benefit of adjuvant chemotherapy in patients with estrogen receptor (ER)+, HER2- early breast cancer who receive 5 years of endocrine therapy. The feasibility of Oncotype DX® testing with diagnostic core needle biopsies (CNB) was demonstrated through analysis of a large commercial laboratory experience. The neoadjuvant ADAPT study showed the clinical utility of deriving the Recurrence Score® (RS) result from CNB as well. However, data are limited on concordance of RS results from paired CNB and surgical samples. Our primary objective was to determine the degree of concordance of RS results between paired tissue samples from the same primary breast tumor of patients with early stage, ER+, HER2- invasive breast cancer who had a CNB and subsequent surgical excision (SE; lumpectomy or mastectomy) without intervening systemic therapy. METHODS: Patients with a commercial RS result on either CNB or subsequent SE were identified through medical record and clinical database review. A pathologist reviewed archival tissue samples to confirm eligibility and selected the other paired CNB or SE block most representative of the tumor tissue for subsequent study Oncotype DX testing. Descriptive statistics were used to summarize patient and tumor characteristics. Wilcoxon signed rank, McNemar’s and Cochran-Mantel-Haenszel tests were used to compare RS between SE and CNB paired samples. Cohen’s Kappa and 95% confidence intervals (CIs) were used to compare the agreement of categorical RS (0-25 and 26-100) result between SE and CNB paired samples. Lin’s concordance correlation and 95% CIs were used to compare agreement of continuous RS between paired samples. RESULTS: A total of 134 patients were identified with paired CNB and SE samples with a median time of 34 (range 5-103) days between collections. Of the 134 patients, a commercial RS result was available for 25 (18.7%) with CNB and 109 (81.3%) with SE. A study RS result was then generated on the other paired sample. Median age was 62 years (range 33-99; 23 patients [17%] were age ≤50 vs. 111 [83%] age &amp;gt;50). Cases were predominantly node negative (107/134 node negative vs. 26/134 node positive; 1/134 unknown) invasive ductal carcinomas (104/134 ductal vs. 18/134 classic lobular vs. 12/134 other). Mean RS results were 15.6 (range 0-56) and 16.6 (range 0-59) for CNB and SE, respectively (p = 0.003), with no statistically significant differences in RS category defined as low vs. high risk (119/134 CNB vs. 114/134 SE for RS 0-25 and 15/134 CNB vs 20/134 SE for RS 26-100; p = 0.13) or low vs. intermediate vs. high risk (85/134 CNB vs. 78/134 SE for RS 0-17 and 43/134 CNB vs. 48/134 SE for RS 18-30 and 6/134 CNB vs. 8/134 SE for RS 31-100; p = 0.19). No differences were observed based on age relative to 50 years or nodal status (data not shown). Cohen’s Kappa statistic k = 0.64 (95% CI, 0.44-0.83) supports substantial agreement between the CNB and SE samples. Lin’s concordance correlation coefficient (CCC) demonstrates similar agreement for the continuous RS result between paired samples (CCC = 0.86 [0.80-0.90]). Overall percent agreement was 91.8% (87.1-96.4%). CONCLUSIONS: Studies evaluating the level of concordance of ER, progesterone receptor, and HER2 biomarker status between CNB and SE samples have found high concordance rates, albeit varied. We now provide data demonstrating high concordance of RS results between CNB and SE specimens obtained from the same breast primary tumor without intervening systemic therapy. This supports use of either biopsy source for testing with a genomic predictor to identify those patients who will most likely benefit from chemotherapy and which patients may be spared. Citation Format: Aziza Nassar, Jodi Carter, Paige Innis, Satish Seerapu, Christy Russell, Helena Hanna, Abigail Lochala, Minetta Liu. Concordance analysis of paired breast cancer core needle biopsies and surgical excision samples using the Oncotype DX Breast Recurrence Score® assay [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-08-09.

GNPNAT1 is a Biomarker That Predicts a Poor Prognosis of Breast Cancer.

Breast cancer (BC) is increasingly becoming the primary reason for death in women, which sounded the alarm. Thus, finding a novel management target for BC is imminent. The data on gene expression and clinicopathological characteristics were downloaded from The Cancer Genome Atlas (TCGA). The expression of GNPNAT1 in 40 paired breast cancer and adjacent tissues was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Univariate and Multivariate logistic regression methodology was applied to analyze the prognostic factors for lymph node metastasis (LNM). Based on the status of breast cancer-relative receptors, patients were distributed into six groups, and then the Kaplan-Meier survival analysis with a Log rank test was applied to investigate the involvement among the expression of GNPNAT1 and overall survival (OS). We found higher expression of GNPNAT1 was connected with poor survival in breast cancer by COX regulation analysis. GO, KEGG, and GSEA analysis prompted that GNPNAT1 was connected with the defense mechanism of cells, cell proliferation, and division. Immunization infiltration analysis showed that high GNPNAT1 was negatively connected with 16 immunization infiltration cell types and positively connected with four immunization infiltration cell types. As a whole, our results indicated that GNPNAT1 might be a probable biomarker for diagnosis and prognosis in breast cancer.

A group of new hypermethylated long non-coding RNA genes associated with the development and progression of breast cancer

Breast cancer is the most common type of cancer among women. The study of the mechanisms of metastasis, the main cause of death from breast cancer, as well as the search for new markers for early diagnosis and prognosis of breast cancer is an extremely topical issue. New perspectives in the diagnosis and treatment of breast cancer are opened by the mechanisms of gene regulation involving non-coding RNAs, in particular, long non-coding RNAs (lncRNAs). In this work, we analyzed the methylation level of seven lncRNA genes (MEG3, SEMA3B-AS1, HAND2-AS1, KCNK15-AS1, ZNF667-AS1, MAGI2-AS3, and PLUT) by quantitative methyl-specific PCR on a set of 79 paired (tumor/normal) samples breast cancer. Hypermethylation of all seven lncRNA genes was revealed, and hypermethylation of HAND2-AS1, KCNK15-AS1, MAGI2-AS3 and PLUT was detected by us in breast cancer for the first time. It was found that the level of methylation of the studied lncRNA genes correlated statistically significantly with the stage of the tumor process, the size of the tumor, and the presence of metastases in the lymph nodes. Thus, methylation of the seven studied lncRNA genes is associated with the development and progression of breast cancer, and these genes can be useful as potential markers in the diagnosis and prognosis of breast cancer.

Downregulation of long noncoding RNA B4GALT1-AS1 is associated with breast cancer development

The misregulation of long non-coding RNAs (lncRNAs) is related to the progressive evolution of various human cancers, such as Breast cancer (BC). The role of lncRNA B4GALT1-AS1 has been investigated in some human cancers. Therefore, studying B4GALT1-AS1 expression was aimed for the first time in the tumor and marginal tissues of BC in this study. The cancer genome atlas (TCGA) database was utilized to evaluate the relative expression of B4GALT1-AS1 in BC and other cancers. RNA was extracted from twenty-eight paired BC and marginal tissues, and cDNA was synthesized. The quantitative expression level of B4GALT1-AS1 was evaluated using real-time PCR. The bioinformatics analyses were performed to identify co-expression genes and related pathways. B4GALT1-AS1 was significantly downregulated in BC specimens compared to tumor marginal samples. The TCGA data analysis confirmed the downregulation of B4GALT1-AS1 in BC. The bioinformatics analysis discovered the correlation between 700 genes and B4GALT1-AS1 and identified GNAI1 as the high degree gene which was positively correlated with B4GALT1-AS1 expression. It seems B4GALT1-AS1 provides its function, at least partly, in association with one of the hippo pathway components, YAP, in other cancers. This protein has the opposite role in BC and its loss of function can result in poor survival in BC. Further research is needed to investigate the interaction between B4GALT1‐AS1 and YAP in various subtypes of BC.

Expression analysis of PPAR-related lncRNAs in breast cancer

Breast cancer is a genetically heterogeneous disorder associated with dysregulation of several genes. Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor that is expressed by many tumoral cells such as transformed breast cancer cells. We investigated expressions of nine PPARγ-related lncRNAs, namely KCNIP2-AS1, TRHDE-AS1, FAM13A-AS1, ALDH1A1-AS2, SH3BP5-AS1, HID1-AS1, LINC01140, LIPE-AS1 and ABCA9-AS1 in paired breast cancer samples and non-tumoral tissues. Expression assays showed lower expression levels of TRHDE-AS1, ALDH1L1-AS2, KCNIP2-AS1, ABCA9-AS1, LIPE-AS1 and LINC01140 in tumoral compared with non-tumoral samples. The mentioned genes could differentiate between breast tumors and non-tumoral samples with AUC values ranging from 0.77 to 0.62 for LINC01140 and LIPE-AS1, respectively. The highest specificity and sensitivity values were reported for KCNIP2-AS1 and LINC01140, respectively. Significant correlations were reported between all pairs of genes in both tumoral and non-tumoral tissues. The most robust ones were between ABCA9-AS1 and KCNIP2-AS1 (correlation coefficient=0.85) in non-tumoral tissues and between LIPE-AS1 and TRHDE-AS1 (correlation coefficient=0.83) in tumoral tissues. There was a significant negative association between expression levels of KCNIP2-AS1 gene in tumor tissues and different histological grades. Besides, there was a significant negative association between expression levels of FAM13A-AS1, KCNIP2-AS1and LIPE-AS1 genes in tumor tissues and different mitotic rates. Taken together, PPARγ-related lncRNAs might be regarded as potential contributors to the pathogenesis of breast cancer.

An insight into the potential role of LINC00968 in luminal breast cancer: Case-control study and bioinformatics analysis

BackgroundLuminal A and B subtypes of breast cancer (BC) comprises up to 70% of all BC patients. LncRNAs can affect many biological and pathological processes, and dysregulation of them is related to human cancers. The potential role of lncRNA LINC00968 in luminal BC is still unclear. Materials and methodsWe analyzed the LINC00968 expression across 44 paired luminal BC tissues from the TCGA-BRCA RNA sequencing dataset. Besides, we used the GEPIA2 web server and GENEVESTIGATOR software, as well. Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) assay was performed to confirm the LINC00968 expression in 71 paired luminal BC tissues and two luminal A cell lines (MCF7 and T47D). Moreover, to better understanding the potential role of LINC00968 in luminal BC, computational data analyses including co-expression analysis, functional annotation analysis, and genetic alteration analysis have been done. ResultsThe results of data analyses retrieved from BRCA dataset and databases revealed the significant downregulation of LINC00968 in luminal A and B BC. Also, the results of qRT-PCR in luminal BC tissues and cell lines confirmed the earlier data. LINC00968 expression was negatively associated with tumor stage and lymph node metastasis. Additionally, functional annotation analyses revealed that LINC00968 might be involved in vascular development and angiogenesis, extracellular matrix organization, and cell motility and migration. LINC00968 might play role in some cancer-related signaling pathways. ConclusionOur study found that downregulation of LINC00968 might promote tumorigenesis, invasion, and metastasis of luminal BC.

IGJ suppresses breast cancer growth and metastasis by inhibiting EMT via the NF‑κB signaling pathway.

Breast cancer metastasis is the primary cause of mortality of patients with breast cancer. The present study aimed to explore the role and underlying mechanisms of IGJ in the invasion and metastasis of breast cancer. The Cancer Genome Atlas database was utilized to analyze the differential gene expression profiles in patients with breast cancer with or without metastasis; the target gene, joining chain of multimeric IgA and IgM (JCHAIN, also known as IGJ, as referred to herein), with significant expression and with prognostic value was screened. The expression levels of IGJ in human breast cancer paired tissues and cell lines were detected using reverse transcription‑quantitative PCR and western blot analysis. IGJ differential expression was detected in paired human breast cancer tissues using immunohistochemistry. The role of IGJ in breast cancer was verified using CCK‑8, invasion and migration assays, and scratch tests invivo and invitro. Further exploration of the role and mechanism of IGJ in breast cancer was conducted through Gene Set Enrichment Analysis, Kyoto Encyclopedia of Genes and Genomes analysis, western blot analysis and immunofluorescence experiments. Through the analysis of gene expression profiles, it was found that IGJ was poorly expressed in patients with breast cancer with metastasis compared to patients with non‑metastatic breast cancer. The overexpression of IGJ was associated with an improved distant metastasis‑free survival and overall survival (OS). COX multivariate regression analysis demonstrated that IGJ was an independent prognostic factor for the OS and relapse‑free survival of patients with breast cancer. In comparison to healthy breast cancer adjacent tissues and cell lines, IGJ was poorly expressed in breast cancer tissues and cell lines (P<0.05). Further analyses indicated that the overexpression of IGJ suppressed the proliferation, invasion and metastasis of breast cancer cells in vivo and invitro by inhibiting the occurrence of epithelial‑to‑mesenchymal transition (EMT) and suppressing the nuclear translocation of p65. Finally, rescue experiments indicated that IGJ restricted the proliferation and metastasis of breast cancer cells by regulating the NF‑κB signaling pathway. On the whole, the present study demonstrates that IGJ suppresses the invasion and metastasis of breast cancer by inhibiting both the occurrence of EMT and the NF‑κB signaling pathway. These findings may provide novel biomarkers and potential therapeutic targets for the treatment of metastatic breast cancer.

The dynamics of HER2-low expression during breast cancer progression.

Low HER2 expression is emerging as an actionable target for the treatment of breast cancer (BC) with the antibody drug conjugate Trastuzumab deruxtecan. The aim of the study was to characterize the dynamics of HER2 expression during BC progression. We evaluated the evolution of HER2 expression in 171 paired primary and metastatic BCs (pBCs/mBCs) by including the HER2-low category. The proportions of HER2-low cases were 25.7% in pBCs and 23.4% in mBCs, respectively, while those of HER2-0 cases were 35.1% and 42.7%, respectively. The overall conversion rate between HER2-0 and HER2-low was 31.7%. HER2-low switching to HER2-0 was more frequent than the reverse (43.2% vs. 23.3%; P = 0.03). Two (3.3%) and 9 (20.5%) cases of pBCs with a HER2-0 and a HER2-low status, respectively, were converted to HER2-positive mBCs. In contrast, 10 (14.9%) HER2-positive pBCs were converted to HER2-0 and an identical number to HER2-low mBCs, respectively, significantly higher than that when compared to the HER2-0 to HER2-positive (P = 0.03), but not HER2-low to HER2-positive conversion. No significant difference was found when comparing the conversion rates among the common organs of relapse. Of the 17 patients with multiorgan metastases, 41.2% had discordance among the different sites of relapse. HER2-low BCs constitute a heterogeneous group of tumors. Low HER2 expression is dynamic, with significant discordance between primary tumors and advanced disease as well as the distant sites of relapse. Repeat biomarker studies from advanced disease are warranted in making appropriate treatment plans in the pursuit of precision medicine.

Gene expression profiling of paired primary breast cancers and brain metastases to identify brain metastases-specific biological changes.

1032 Background: Despite the clinical impact of breast cancer (BC) brain metastases (BM), their biological complexity still remains poorly understood. We here evaluate the genomic profile of paired primary and metastatic samples to characterize biological changes acquired by BC during metastatization to the brain. Methods: The expression of 758 BC–related genes was evaluated using the BC360 Panel (nCounter platform) in matched primary BC and their associated BM. Intrinsic molecular subtyping was determined using the PAM50 subtype predictor (Parker et al. JCO 2009). Hormone receptor (HR) and HER2 status were evaluated on the BCBM. A False Discovery Rate (FDR) corrected paired two-class SAM was used to identify changes in single genes expression between paired BC and BMs samples. Results: Twenty-one matched primary BC and BMs samples were analyzed. BC subtype evaluated on the BMs was distributed as follows: 24% (N=5) HR-/HER2-, 24% (N=5) HR+/HER2- and 52% (N=11) HER2+. A considerable shift in PAM50 subtype was observed between primary BC and paired BM. While subtype concordance was high for HER2-enriched (HER2-E) tumors (100%), all Luminal A (LumA) primary BCs switched to either HER2-E (75%) or Luminal B (LumB, 25%) on the BM sample, as did 45% of Basal-like BCs (Basal to HER2-E). A clinical switch from HER2- primary BC to HER2+ BM (defined by IHC and ISH) was observed in only one pair (5%). Consistently, HER2-E (p&lt;0.001) and LumB (p=0.001) PAM50 signatures were significantly more expressed, while the LumA (p&lt;0.001) and Normal-like (p=0.001) signatures were significantly less expressed in BMs as compared to paired primary BCs (FDR-corrected Wilcoxon paired signed rank test). No significant change was observed in the expression of the Basal-like signature (p=0.562). Among the 758 evaluated genes, 60 and 318 genes, respectively, were significantly up- and downregulated in BMs as compared to primary BC (FDR&lt;5%). Upregulated genes were enriched, among others, in genes involved in survival and migration (e.g. FGFR4), cell proliferation (e.g. CCNB1, CDK1) and HER2-amplicon (e.g. ERBB2, GRB7). Downregulated genes were enriched in genes involved in immune response (e.g. CD8A, CCL5, PDCD1LG2, TNF), angiogenesis (e.g. PDGFRB) and response to hormonal stimuli (e.g. ESR1, BCL2). Conclusions: Gene-expression profiling of matched primary BCs and BMs shows recurrent gene expression modifications in metastatic samples which might have potential therapeutic implications (e.g. acquisition of HER2-E subtyping and increase in ERBB2 mRNA levels). [Table: see text]

Hsa_circ_0060467 promotes breast cancer liver metastasis by complexing with eIF4A3 and sponging miR-1205

Breast cancer (BC) is the most common cancer and the top cause of female mortality worldwide. The prognosis for patients with breast cancer liver metastasis (BCLM) remains poor. Emerging studies suggest that circular RNAs (circRNAs) are associated with the progression of BC. Exploration of circRNAs presents a promising avenue for identifying metastasis-targeting agents and improving the prognosis of patients with BCLM. Microarray and bioinformatic analyses were used to analyze differentially expressed circRNAs between three pairs of BCLM and primary BC. The roles of hsa_circ_0060467 (circMYBL2) and its target gene E2F1 in BC cells were explored by multiple functional experiments. And xenograft mouse models and hepatic metastases of BC hemi-spleen models were used to illustrate the function of circMYBL2 in vivo. The intrinsic molecular mechanism involving circMYBL2 was confirmed by bioinformatics analyses, RIP assays, CHIRP assays, luciferase reporter assays, and rescue experiments. CircMYBL2 was overexpressed in BCLM tissues and BC cells. Functionally, circMYBL2 can facilitate the proliferation and liver metastasis of BC. Mechanistically, circMYBL2 upregulated the transcription factor E2F1 by sponging miR-1205 and complexing with eukaryotic translation initiation factor 4A3 (eIF4A3) and then facilitated the epithelial-mesenchymal transition (EMT) process in BC cells. Our findings showed that circMYBL2 promoted the tumorigenesis and aggressiveness of BC through the circMYBL2/miR-1205/E2F1 and circMYBL2/eIF4A3/E2F1 axes, which may provide a novel targeted therapy for patients with BCLM.

Breast Cancer Prognostic Hub Genes Identified by Integrated Transcriptomic and Weighted Network Analysis: A Road Toward Personalized Medicine.

Breast cancer (BC) is the second-most common type and among the leading causes of worldwide cancer-related deaths. There is marked person-to-person variability in susceptibility to, and phenotypic expression and prognosis of BC, a predicament that calls for personalized medicine and individually tailored therapeutics. In this study, we report new observations on prognostic hub genes and key pathways involved in BC. We used the data set GSE109169, comprising 25 pairs of BC and adjacent normal tissues. Using a high-throughput transcriptomic approach, we selected data on 293 differentially expressed genes to establish a weighted gene coexpression network. We identified three age-linked modules where the light-gray module strongly correlated with BC. Based on the gene significance and module membership features, peptidase inhibitor 15 (PI15) and KRT5 were identified as our hub genes from the light-gray module. These genes were further verified at transcriptional and translational levels across 25 pairs of BC and adjacent normal tissues. Their promoter methylation profiles were assessed based on various clinical parameters. In addition, these hub genes were used for Kaplan-Meier survival analysis, and their correlation with tumor-infiltrating immune cells was investigated. We found that PI15 and KRT5 may be potential biomarkers and potential drug targets. These findings call for future research in a larger sample size, which could inform diagnosis and clinical management of BC, thus paving the way toward personalized medicine.

The correlation of ESR1 genetic aberrations with estrogen receptor and progesterone receptor status in metastatic and primary estrogen receptor-positive breast carcinomas

Genetic aberrations in the Estrogen Receptor 1 (ESR1) gene have been identified as an important mechanism of resistance to endocrine therapy in metastatic breast carcinoma. In this study, we aimed to correlate ESR1 genetic aberrations with the ER and PR status in paired metastatic and primary breast carcinomas. Patients with ER-positive breast cancer were divided into two groups: ESR1 genetic aberration (n=26) and wild-type control (n=29) based on genetic profiling of their metastatic tumors. Clinicopathological features and ER/PR status were analyzed in paired primary and metastatic tumors. Although there was no significant difference in ER expression between the ESR1 aberration and control groups in primary tumors, ER positivity rate in metastatic tumors was significantly higher in the ESR1 aberration group than in the control group (100% vs. 86%, P<.05). ESR1 aberrated cases were associated with more liver metastases than control tumors (46% vs. 10%, P<.01). The ER percentage and intensity slightly increased from primary to metastatic tumors in the ESR1 aberration group compared to a decrease in both in the wild-type group (percentage increase 2% vs. decrease 19%, P=.0594; intensity increase 0.04 vs. decrease 0.8, p<.05). Patients with ESR1 aberrated metastases were more likely than those with wild-type ESR1 metastases to have the following characteristics: 1) ER percentage ≥90% and intensity >2, as well as PR percentage ≥30% and intensity >1 in metastatic tumors; 2) ER percentage ≥90% and PR percentage ≥70% in primary tumors; and 3) slightly increase in ER percentage and intensity from primary to metastatic tumors. Based on the ER/PR parameters of paired primary and metastatic breast cancer, ESR1 aberration in metastasis may be predicted.

Proteomics analysis of human breast milk by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) coupled with mass spectrometry to assess breast cancer risk.

Breast cancer (BC) is one of the most common cancers and one of the most common causes for cancer-related mortality. Discovery of protein biomarkers associated with cancer is considered important for early diagnosis and prediction of the cancer risk. Protein biomarkers could be investigated by large-scale protein investigation or proteomics, using mass spectrometry (MS)-based techniques. Our group applies MS-based proteomics to study the protein pattern in human breast milk from women with BC and controls and investigates the alterations and dysregulations of breast milk proteins in comparison pairs of BC versus control. These dysregulated proteins might be considered potential future biomarkers of BC. Identification of potential biomarkers in breast milk may benefit young women without BC, but who could collect the milk for future assessment of BC risk. Previously we identified several dysregulated proteins in different sets of human breast milk samples from BC patients and controls using gel-based protein separation coupled with MS. Here, we performed 2D-PAGE coupled with nano-liquid chromatography-tandem MS (nanoLC-MS/MS) in a small-scale study on a set of six human breast milk pairs (three BC samples vs. three controls) and we identified several dysregulated proteins that have potential roles in cancer progression and might be considered potential BC biomarkers in the future.

Comprehensive proteome, phosphoproteome and kinome characterization of luminal A breast cancer.

Breast cancer is one of the most frequently occurring malignant cancers worldwide. Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the two most common histological subtypes of breast cancer. In this study, we aimed to deeply explore molecular characteristics and the relationship between IDC and ILC subtypes in luminal A subgroup of breast cancer using comprehensive proteomics and phosphoproteomics analysis. Cancer tissues and noncancerous adjacent tissues (NATs) with the luminal A subtype (ER- and PR-positive, HER2-negative) were obtained from paired IDC and ILC patients respectively. Label-free quantitative proteomics and phosphoproteomics methods were used to detect differential proteins and the phosphorylation status between 10 paired breast cancer and NATs. Then, the difference in protein expression and its phosphorylation between IDC and ILC subtypes were explored. Meanwhile, the activation of kinases and their substrates was also revealed by Kinase-Substrate Enrichment Analysis (KSEA). In the luminal A breast cancer, a total of 5,044 high-confidence proteins and 3,808 phosphoproteins were identified from 10 paired tissues. The protein phosphorylation level in ILC tissues was higher than that in IDC tissues. Histone H1.10 was significantly increased in IDC but decreased in ILC, Conversely, complement C4-B and Crk-like protein were significantly decreased in IDC but increased in ILC. Moreover, the increased protein expression of Septin-2, Septin-9, Heterogeneous nuclear ribonucleoprotein A1 and Kinectin but reduce of their phosphorylation could clearly distinguish IDC from ILC. In addition, IDC was primarily related to energy metabolism and MAPK pathway, while ILC was more closely involved in the AMPK and p53/p21 pathways. Furthermore, the kinomes in IDC were primarily significantly activated in the CMGC groups. Our research provides insights into the molecular characterization of IDC and ILC and contributes to discovering novel targets for further drug development and targeted treatment.

Comparative Genomic Profiling of Second Breast Cancers following First Ipsilateral Hormone Receptor-Positive Breast Cancers.

We compared the mutational profile of second breast cancers (SBC) following first ipislateral hormone receptor-positive breast cancers of patient-matched tumors to distinguish new primaries from true recurrences. Targeted next-generation sequencing using the Oncomine Tumor Mutation Load Assay. Variants were filtered according to their allele frequency ≥ 5%, read count ≥ 5X, and genomic effect and annotation. Whole genome comparative genomic hybridization array (CGH) was also performed to evaluate clonality. Among the 131 eligible patients, 96 paired first breast cancer (FBC) and SBC were successfully sequenced and analyzed. Unshared variants specific to the FBC and SBC were identified in 71.9% and 61.5%, respectively. Paired samples exhibited similar frequency of gene variants, median number of variants per sample, and variant allele frequency of the reported variants except for GATA3. Among the 30 most frequent gene alterations, ARIDIA, NSD2, and SETD2 had statistically significant discordance rates in paired samples. Seventeen paired samples (17.7%) exhibited common variants and were considered true recurrences; these patients had a trend for less favorable survival outcomes. Among the 8 patients with available tissue for CGH analysis and considered new primaries by comparison of the mutation profiles, 4 patients had clonally related tumors. Patient-matched FBC and SBC analysis revealed that only a minority of patients exhibited common gene variants between the first and second tumor. Further analysis using larger cohorts, preferably using single-cell analyses to account for clonality, might better select patients with true recurrences and thereby better inform the decision-making process.

Down-regulation of SLC16A-AS1 and LINC00900 lncRNAs in Iranian patients with breast cancer.

Long non-coding RNAs (lncRNAs) influence pathetiology of breast cancer. Besides, VDR and ESR1 signaling pathways are two important pathways in this malignancy. In the present mixed bioinformatics and expression assay study, we have identified lncRNAs that are co-expressed with VDR and ESR1 in breast cancer tissues and analyzed their expression in 42 paired breast cancer and non-cancerous specimens. Expression of SLC16A-AS1 was significantly lower in breast cancer tissues compared with paired non-cancerous samples (expression ratio = 0.27, P value < 0.001). Similarly, LINC00900 was down-regulated in cancer tissues compared with non-cancerous ones (expression ratio = 0.26, P value = 0.01). There were no significant differences in the expressions of VDR and AATBC between these two sets of samples. Expression levels of VDR and AATBC were associated with histological grade (P values = 0.02 and 0.03, respectively). Moreover, expression of VDR was associated with tumor size (Pvalue = 0.02). Finally, expression levels of SLC16A-AS1 were associated with first pregnancy age (P value = 0.006). In brief, the results of current study further support involvement of VDR and ESR1-associated lncRNAs in breast cancer.

Mitochondrial calcium uniporter activates TFEB-driven autophagy to promote migration of breast cancer cells.

Tumor metastasis is the leading cause of death in breast cancer (BC) patients and is a complicated process. Mitochondrial calcium uniporter (MCU), a selective channel responsible for mitochondrial Ca2+ uptake, has been reported to be associated with tumorigenesis and metastasis. The molecular mechanisms of MCU contributing to the migration of BC cells are partially understood. This study investigated the role of MCU in BC cell metastasis and explored the underlying mechanism of MCU-mediated autophagy in BC cell migration. The Kaplan-Meier plotter database was used to analyze the prognostic value of MCU mRNA expression. Western blotting was used to examine the expression level of MCU in 4 paired BC and adjacent normal tissues. The cellular migration capability of BC was measured by transwell migration assay and wound healing assay. Western blotting and reverse transcription-quantitative polymerase chain reaction were performed to detect the expression levels of autophagy-related markers. The effects of MCU activation or inhibition on TFEB nuclear translocation in BC cells were detected by laser scanning confocal microscopy. Expression of MCU was found to be negatively correlated with BC patient prognosis in the Kaplan-Meier plotter database. Compared with the adjacent normal tissues, MCU was markedly up-regulated in the BC tissues. MCU overexpression promoted cellular migration, activated autophagy, and increased TFEB nuclear translocation in BC cells, whereas its knockdown produced the opposite effects. MCU activates TFEB-driven autophagy to promote BC cell metastasis and provides a potential novel therapeutic target for BC clinical intervention.