Metastatic Breast Cancer Tissues Research Articles

Phase Ib pharmacodynamic study of the MNK inhibitor Tomivosertib (eFT508) combined with paclitaxel in patients with refractory metastatic breast cancer.

Preclinical data motivate clinical evaluation of inhibitors of mitogen-activated protein kinase-interacting kinases 1 and 2 (MNK1/2). We conducted a phase 1b clinical trial to study target engagement and safety of tomivosertib, a MNK1/2 inhibitor, alone and in combination with paclitaxel. Eligible patients had metastatic breast cancer resistant to standard of care treatments. Biopsies were obtained at baseline and during treatment with tomivosertib, and then tomivosertib was continued with addition of paclitaxel until disease progression or toxicity. Serum drug levels were measured, and pharmacodynamic endpoints included immunohistochemistry, proteomics, translatomics, and imaging mass cytometry. Tomivosertib alone and in combination with paclitaxel was well tolerated. There was no pharmacokinetic interaction between the drugs. We observed a clear reduction in phosphorylation of eIF4E at S209, a major substrate of MNK1/2, and identified tomivosertib-induced perturbations in the proteome, translatome, and cellular populations of biopsied metastatic breast cancer tissue. We conclude that tomivosertib effectively inhibits MNK1/2 activity in metastatic breast cancer tissue, and that it can safely be combined with paclitaxel in future phase II studies. We demonstrate feasibility of using proteomic profiles, translatomic profiles, and spatial distribution of immune cell infiltrates for clinical pharmacodynamic studies.

RNA-binding protein LSM7 facilitates breast cancer metastasis through mediating alternative splicing of CD44

AimsThe RNA-binding protein LSM7 is essential for RNA splicing, acting as a key component of the spliceosome complex; however, its specific role in breast cancer (BC) has not been extensively investigated. Materials and methodsLSM7 expression in BC samples was evaluated through bioinformatics analysis and immunohistochemistry. The impact of LSM7 on promoting metastatic tumor characteristics was examined using transwell and wound healing assays, as well as an orthotopic xenograft model. Additionally, the involvement of LSM7 in alternative splicing of CD44 was explored via RNA immunoprecipitation and third-generation sequencing. The regulatory role of TCF3 in modulating LSM7 gene expression was further elucidated using luciferase reporter assays and chromatin immunoprecipitation. Key findingsOur findings demonstrate that LSM7 was significantly overexpressed in metastatic BC tissues and was associated with poor prognostic outcomes in patients with BC. LSM7 overexpression markedly increased the migratory and invasive capabilities of BC cells in vitro and significantly promoted spontaneous lung metastasis in vivo. Furthermore, RIP-seq analysis revealed that LSM7 binded to CD44 RNA, enhancing the expression of its alternatively spliced isoform CD44s, thereby driving BC metastasis and invasion. Additionally, the transcription factor TCF3 was found to activate LSM7 transcription by directly binding to its promoter. SignificanceIn summary, this study highlights the pivotal role of LSM7 in the production of the CD44s isoform and the promotion of breast cancer metastasis. Targeting the TCF3/LSM7/CD44s axis may offer a promising therapeutic strategy for breast cancer treatment.

Abstract PO5-14-10: Dynamics and prognostic value of subtype specific TROP2 expression in matched pair samples of primary (PBC) and metastatic breast cancer (MBC) tissues

Abstract Background: The genotype and phenotype of breast cancer may change during disease progression. This is of particular interest in the advent of targeted treatment adressing extracellular targets such as TROP2, which serves as chemotherapy carrier rather than being required for tumor cell survival. In addition, technical issues may affect biomarker assessment when comparing primary breast cancer (PBC) tissues with biopsies of metastatic breast cancer (MBC) tissues at distant sites. Here we analyzed TROP2 expression on mRNA level by RT-qPCR in primary tumor and metastatic tissues on basis of molecular subtyping to determine subtype specificity and target gene stability/dynamics. Methods: The tumor bank of a single institution was screened for paraffin-embedded pairs of PBC and MBC tissue samples and a total of 34 matched PBC and MBC pairs were retrieved. RNA was extracted using a bead-based extraction method (RNXtract® IVD kits, Cerca Biotech). Multiplex RT-qPCR was performed using TaqMan® based primer probe sets for ESR1/PGR/ERBB2 and MKI67 (MammaTyper® IVD kits, Cerca Biotech) as well as TROP2 (TargetTyper RUO kits, STRATIFYER Molecular Pathology GmbH). Correlation analysis, Scatter Plots, Partitioning tests and Kaplan Meier analysis were performed using the SAS JMP® 9.0.0 software. Results: Samples from 34 patients with MBC and PBC were available. Positivity of PBC RT-qPCR for ESR1, PGR and HER2 was 78%, 70% and 3%. The overall agreement between matched primary and metastatic lesions 90%, 70% and 100% for RT-qPCR of ESR1, PGR and HER2. Median expression of TROP2 was 2 fold lower in metastatic tissues compared to matched primary tumor tissues, while the lower quartile of mRNA expression dropped 4 fold. In primary tumor tissues TROP2 expression was significantly associated with PGR and HER2 expression (Spearman r=0.5284 p=0.0013 and r=0.3950 p=0.0208; respectively), while no significant association was found for ESR1 and MKI67. In contrast, no significant association with neither PGR nor HER2 was found (Spearman r=0.0461 p=0.8041 and r=0.0461 p=0.7955; respectively), while TROP2 trended to be associated with MKI67 in metastatic lesions (Spearman r=0.3247 p=0.0610). In line with the strong positive association of TROP2 with PGR in primary tumor tissues, elevated TROP2 levels were associated with substantially longer median distant metastasis free survival of 72 months, while TROP2 negative tumors exhibited a median DDFS of only 24 months (p=0,0091). In contrast, no prognostic value for TROP2 mRNA level could be determined from metastatic lesions and determining the time from initial metastasis until death. Conclusion: In this selected matched pair, metastatic cohort, TROP2 mRNA in primary tumors is strongly associated with luminal type of breast cancer expressing higher levels of PGR mRNA, while no such correlation was found in matched metastatic tissues. Moreover, TROP2 is significantly downregulated in metastatic lesions. As TROP2 is expressed in luminal tumors, it may serve as a valuable target for luminal tumors of higher progression risk. However, TROP2 expression is unstable and requires careful decision making on actual biopsies rather than archival tissue samples. Citation Format: Thomas Deutsch, Ralph Wirtz, Stefan Stefanovic, Andreas Hartkopf, Hans-Peter Sinn, Andreas Schneeweiss, Markus Wallwiener. Dynamics and prognostic value of subtype specific TROP2 expression in matched pair samples of primary (PBC) and metastatic breast cancer (MBC) tissues [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 PO5-14-10.

Abstract PO2-27-06: ANXA9 facilitates the exocrine secretion of S100A4 and promotes pleural metastasis in breast cancer

Abstract Metastasis to distant organs stands as the primary contributor to mortality in cases of breast cancer. While localized breast cancer boasts a 5-year survival rate of roughly 99%, this figure drastically declines to approximately 26% for patients afflicted with distant metastasis. Regrettably, the current therapeutic landscape lacks efficacious and targeted interventions for the treatment of metastatic breast cancer. Our investigation has revealed a significant upregulation of S100A4 in metastatic breast cancer tissues, with notably high expression observed in the malignant pleural effusion of patients in advanced stages of the disease. Upon acceptance of the effective treatment, the expression of S100A4 in malignant pleural effusion decreased, along with a decrease in several cytokines, including IL-6, IL-8, CCL2, and CCL5. The breast cancer cellular models demonstrated that ANXA9 has the ability to regulate the excretion of S100A4 from cells into the surrounding medium. Similarly, the mice model of pleural metastasis indicated that the downregulation of ANXA9 (sh-ANXA9) resulted in decreased levels of S100A4 and cytokines IL-6, IL-8, CCL2, and CCL5 in the mice's hydrothorax. Additionally, our findings suggest that ANXA9 regulates the excretion of S100A4 through phosphorylation of ANXA9. The cellular membrane harbored phosphorylated ANXA9, which exhibited two phosphorylation sites. Upon mutation of these sites, the level of ANXA9 phosphorylation decreased, leading to a reduction in the exocrine release of ANXA9 from cells into the medium. Our research further revealed the significant involvement of ANXA9 in facilitating the progression of breast cancer, thereby suggesting that therapeutic interventions targeting ANXA9 could prove efficacious in treating metastatic breast cancer. This work was supported by Shanghai Sailing Program to Dengfeng Li (No. 20YF1437800), grant from the National Natural Science Foundation of China to Dengfeng li (No. 82103454) and grant from the National Natural Science Foundation of China to Lin Fang (No. 82073204). Citation Format: Xiqian Zhou, Dengfeng Li, Lin Fang, Junyong Zhao. ANXA9 facilitates the exocrine secretion of S100A4 and promotes pleural metastasis in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-27-06.

ANXA9 facilitates S100A4 and promotes breast cancer progression through modulating STAT3 pathway

Breast cancer has the highest global incidence and mortality rates among all cancer types. Abnormal expression of the Annexin family has been observed in different malignant tumors, including upregulated ANXA9 in breast cancer. We found highly expressed ANXA9 in metastatic breast cancer tissues, which is correlated with breast cancer progression. In vitro, the functional experiments indicated ANXA9 influenced breast cancer proliferation, motility, invasion, and apoptosis; in vivo, downregulation of ANXA9 suppressed breast cancer xenograft tumor growth and lung metastasis. Mechanically, on one side, we found that ANXA9 could mediate S100A4 and therefore regulate AKT/mTOR/STAT3 pathway to participate p53/Bcl-2 apoptosis; on the other side, we found ANXA9 transferred S100A4 from cells into the tumor microenvironment and mediated the excretion of cytokines IL-6, IL-8, CCL2, and CCL5 to participate angiogenesis via self- phosphorylation at site Ser2 and site Thr69. Our findings demonstrate significant involvement of ANXA9 in promoting breast cancer progression, thereby suggesting that therapeutic intervention via targeting ANXA9 may be effective in treating metastatic breast cancer.

Unique macrophage phenotypes activated by BMP signaling in breast cancer bone metastases.

Metastatic breast cancer (mBC) tissue in bone was systematically profiled to define the composition of the tumor microenvironment. Gene expression identified a high myeloid signature of patients with improved survival outcomes. Bone metastases were profiled by spatial proteomics to examine myeloid populations within the stroma that correlated with macrophage functions. Single-cell spatial analysis uncovered macrophage activation in the stroma of mBC bone lesions. Matched BC patient samples of primary breast tumor and bone metastasis tissues were compared for gene expression in the bone, where bone morphogenetic protein 2 (BMP2) was most significantly upregulated. Immune cell changes from breast to bone demonstrated a loss of lymphoid cells but a consistent population of macrophages. BMP-activated macrophages were increased uniquely in bone. Bone marrow-derived macrophage activation coupled with BMP inhibition increased inflammatory responses. Using experimental mouse models of mBC bone metastasis and trained immunity, we found that BMP inhibition restricts progression of metastases early in the macrophage activation state but not after tumors were established in the bone. This study revealed unique myeloid BMP activation states that are distinctly integrated with bone metastases.

Downregulated circPOKE promotes breast cancer metastasis through activation of the USP10-Snail axis.

Breast cancer (BC) is the most commonly diagnosed cancer and the leading cause of cancer-related death among females. Metastasis accounts for the majority of BC related deaths. One feasible strategy to solve this challenging problem is to disrupt the capabilities required for tumor metastasis. Herein, we verified a novel metastasis suppressive circRNA, circPOKE in BC. circPOKE was downregulated in primary and metastatic BC tissues and overexpression of circPOKE inhibited the metastatic potential but not the proliferative ability of BC cells in vitro and in vivo. Mechanistically, circPOKE competitively binds to USP10, and reduces its binding to Snail, a key transcriptional regulator of EMT, thereby inhibiting Snail stability via the protein-ubiquitination degradation pathway. In addition, we found that circPOKE could be secreted into the extracellular space via exosomes and that exosome-carried circPOKE significantly inhibited the invasive capabilities of BC cells in vitro and in vivo. Furthermore, the levels of circPOKE, USP10 and Snail are clinically relevant in BC, suggesting that circPOKE may be used as a potential therapeutic target for patients with BC metastasis.

Relationship of Ki-67 index in biopsies of metastatic breast cancer tissue and circulating tumor cells (CTCs) at the time of biopsy collection

BackgroundThe proliferation marker Ki-67 is a major pathological feature for the description of the state of disease in breast cancer. It helps to define the molecular subtype and to stratify between therapy regimens in early breast cancer and helps to assess the therapy response. Circulating tumor cells (CTCs) are a negative prognostic biomarker for progression free (PFS) and overall survival (OS) in patients with metastatic breast cancer. Therefore, the CTC count is often described as surrogate for the tumor burden. Both, decrease of Ki-67 and CTC count are considered as evidence for therapy response. The presented work analyzed the correlation between the Ki-67 indices of metastatic tissue biopsies and CTC counts in biopsy time-adjacent peripheral blood samples.Patients and methodsBlood samples from 70 metastatic breast cancer patients were obtained before the start of a new line of systemic therapy. CTCs were enumerated using CellSearch® (Menarini Silicon Biosystems, Bologna, Italy) whereas intact CTCs (iCTCs) and non-intact or apoptotic CTCs (aCTCs) were distinguished using morphologic criteria. The proportion of cells expressing Ki-67 was evaluated using immunohistochemistry on biopsies of metastases obtained concurrently with CTC sampling before the start of a new line of systemic therapy.Results65.7% of patients had a Ki-67 index of > 25%. 28.6% of patients had ≥ 5, 47.1% ≥ 1 iCTCs. 37.1% had ≥ 5, 51.4% ≥ 1 aCTCs. No correlation was shown between Ki-67 index and iCTC and aCTC count (r = 0.05 resp. r = 0.05, Spearman’s correlation index). High CTC-counts did not coincide with high Ki-67 index. High Ki-67, ≥ 5 iCTCs and aCTCs are associated with poor progression free (PFS) and overall survival (OS).ConclusionCTCs and Ki-67 are independent prognostic markers in metastatic breast cancer. High Ki-67 in metastatic tumor tissue is not correlated to high iCTC or aCTC counts in peripheral blood.

Cellular and Molecular Effects of Eribulin in Preclinical Models of Hematologic Neoplasms.

Despite the advances in understanding the biology of hematologic neoplasms which has resulted in the approval of new drugs, the therapeutic options are still scarce for relapsed/refractory patients. Eribulin is a unique microtubule inhibitor that is currently being used in the therapy for metastatic breast cancer and soft tissue tumors. Here, we uncover eribulin's cellular and molecular effects in a molecularly heterogeneous panel of hematologic neoplasms. Eribulin reduced cell viability and clonogenicity and promoted apoptosis and cell cycle arrest. The minimal effects of eribulin observed in the normal leukocytes suggested selectivity for malignant blood cells. In the molecular scenario, eribulin induces DNA damage and apoptosis markers. The ABCB1, ABCC1, p-AKT, p-NFκB, and NFκB levels were associated with responsiveness to eribulin in blood cancer cells, and a resistance eribulin-related target score was constructed. Combining eribulin with elacridar (a P-glycoprotein inhibitor), but not with PDTC (an NFkB inhibitor), increases eribulin-induced apoptosis in leukemia cells. In conclusion, our data indicate that eribulin leads to mitotic catastrophe and cell death in blood cancer cells. The expression and activation of MDR1, PI3K/AKT, and the NFκB-related targets may be biomarkers of the eribulin response, and the combined treatment of eribulin and elacridar may overcome drug resistance in these diseases.

Outcome of Breast Carcinoma between ER, PR with HER-2/Neu Positive Receptor Status

Background: The most frequent kind of cancer among women is breast cancer. Evidence shows that the receptor profile of primary and metastatic breast cancer tissue differs. Archival-paired pathology samples were evaluated to determine the level of hormone receptor discordance. Objective: To evaluate the efficacy of the most frequently used biological indicators in breast cancer for predicting how well a patient would respond to surgical management and their outcome. Materials and Methods: This comparative Study was conducted in the surgery department, Rajshahi Medical College, Bangladesh, with a Matricentred base Study from January 2019 to December 2021. They also included patients (n=100) for whom tissue from either the main or secondary metastatic location was detectable. Cancers were compared and analyzed for their ER, PR, and Her-2/neu status in both the primary and the metastatic. Results: The discordance rate for ER was 17.7% (2-sided p=0.0039), with 9.7% of tumors changing from ER-positive to ER-negative and 8.0% changing from ER-negative to ER-positive. The discordance rate for PR was 37.3% (2-sided p<0.0001), with Each and every one of these tumors going through a transition from PR-positive to PR-negative. As far as Her-2/neu is concerned, no major discrepancies were discovered. Conclusions: Hormone receptor status is significantly different between primary and metastatic breast cancer samples, as suggested by this dataset. Common instances of PR decline were observed. More than half of the patients had a positive hormone status, and it was found that the illness had spread to other organs in most of these cases.

Nuclear import of PTPN18 inhibits breast cancer metastasis mediated by MVP and importin β2

Distant metastasis is the primary cause of breast cancer-associated death. The existing information, such as the precise molecular mechanisms and effective therapeutic strategies targeting metastasis, is insufficient to combat breast cancer. This study demonstrates that the protein tyrosine phosphatase PTPN18 is downregulated in metastatic breast cancer tissues and is associated with better metastasis-free survival. Ectopic expression of PTPN18 inhibits breast cancer cell metastasis. PTPN18 is translocated from the cytoplasm to the nucleus by MVP and importin β2 in breast cancer. Then, nuclear PTPN18 dephosphorylates ETS1 and promotes its degradation. Moreover, nuclear PTPN18 but not cytoplasmic PTPN18 suppresses transforming growth factor-β signaling and epithelial-to-mesenchymal transition by targeting ETS1. Our data highlight PTPN18 as a suppressor of breast cancer metastasis and provide an effective antimetastatic therapeutic strategy.

Quantitative multiplexed analysis of MMP-11 and CD45 in metastatic breast cancer tissues by immunohistochemistry-assisted LA-ICP-MS.

Breast cancer is the leading cause of cancer death in woman and tremendous efforts are undertaken to limit its dissemination and to provide effective treatment. Various histopathological parameters are routinely assessed in breast cancer biopsies to provide valuable diagnostic and prognostic information. MMP-11 and CD45 are tumor-associated antigens and potentially valuable biomarkers for grading aggressiveness and metastatic probability. This paper presents methods for quantitative and multiplexed imaging of MMP-11 and CD45 in breast cancer tissues and investigates their potential for improved cancer characterization and patient stratification. An immunohistochemistry-assisted laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) method was successfully developed and optimized using lanthanide-tagged monoclonal antibodies as proxies to determine spatial distributions and concentrations of the two breast cancer biomarkers. The labeling degree of antibodies was determined via size exclusion-ICP-tandem mass spectrometry (SEC-ICP-MS/MS) employing online calibration via post-column isotope dilution analysis (IDA). The calibration of spatial distributions of labeled lanthanides in tissues was performed by ablating mold-prepared gelatin standards spiked with element standards. Knowledge of labeling degrees enabled the translation of lanthanide concentrations into biomarkers concentrations. The k-means clustering was used to select tissue areas for statistical analysis and mean concentrations were compared for sets of metastatic, non-metastatic and healthy samples. MMP-11 was expressed in stroma surrounding tumor areas, while CD45 was predominantly found inside tumor areas with high cell density. There was no significant correlation between CD45 and metastasis (P=0.70); however, MMP-11 was significantly up-regulated (202%) in metastatic samples compared to non-metastatic (P=0.0077) and healthy tissues (P=0.0087).

Abstract CT521: Phase 1b trial of the MNK1/2 inhibitor tomivosertib combined with paclitaxel in patients with metastatic breast cancer

Abstract MNK is a serine/threonine kinase signaling node in the MAPK pathway, which is phosphorylated and activated by ERK. MNK phosphorylates eIF4E to promote translation of a subset of mRNAs that encode oncogenically relevant proteins including c-Myc, Cyclin D1 and PD-L1. We conducted a phase 1B study [NCT04261218] of tomivosertib (T), an orally administered inhibitor of MNK, to assess target engagement in metastatic breast cancer tissue and tolerability and pharmacokinetics of co-administration with paclitaxel (P). There are no prior reports of co-administration of a MNK inhibitor with a cytotoxic agent. All patients underwent pre-treatment baseline and on-treatment (day ~7) core biopsy of metastatic disease during the T monotherapy lead-in phase; subsequently patients continued oral T 100mg bid (without or with meal) with the addition of IV P at 80mg/m2 day 1 and 8 every 21 days. Results: We report here interim data for the first 14 patients with MBC refractory to standard treatments. Pharmacokinetics: T plasma levels were 21+/- 40 ng/ml throughout the day, and were not influenced by infusion of P. Peak P levels following infusion were 600 +/- 220 ng/ml and fell to below detection limit by 24 hours post infusion, similar to prior reports for single-agent P. Pharmacodynamics: The main endpoint was assessment of T -induced eIF4E phosphorylation in MBC biopsy tissue, estimated by immunostaining with an antibody specific for eIF4E phosphorylated at S209. Phospho-eIF4E staining intensity was generally high in the pre-treatment samples (indicating baseline activation of eIF4E in MBC). In contrast, the on- T biopsies revealed undetectable phopho-eIF4E staining in all patients. These results were confirmed by mass cytometry analysis, which also included a characterization of the immune microenvironment. We performed ribo-seq analysis on 2 mg flash frozen cancer specimens, and this provided preliminary evidence for decreased translation of mRNAs encoding proteins relevant to neoplasia including TMSB4X and MUCL1 on exposure to T. Preliminary analysis of comparison of proteomic data for each patient, comparing baseline and on-drug biopsies, revealed upregulation of proteins involved in fatty acid catabolic processes, consistent with prior experimental work (PMID 32712434). Safety and Clinical: Of 14 initial patients, 1 left the study for adverse reaction (nausea), 1 left study at physician’s discretion, and 5 left the study for progressive disease. There were no indications of increased P toxicity, and T was generally well tolerated in keeping with prior single agent phase I data. Two patients showed a decrease (36% and 45%) in size of metastases, but others showed disease progression, despite evidence for absence of phospho-eIF4E. The results indicate feasibility of a future phase 2 study of the T - P combination. (Supported by a Dream Team grant from Stand Up To Cancer - Canada) Citation Format: Cristiano Ferrario, John Mackey, Karen Gelmon, Poul HB Sorensen, Htoo Zarni Oo, Gregg B. Morin, Sonia del Rincon, Mehdi Amiri, Premal Patel, Harvey W. Smith, Nahum Sonenberg, Michael N. Pollak. Phase 1b trial of the MNK1/2 inhibitor tomivosertib combined with paclitaxel in patients with metastatic breast cancer [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 CT521.

Comprehensive analysis of gene expression profiles to identify differential prognostic factors of primary and metastatic breast cancer

Breast cancer accounts for nearly half of all cancer-related deaths in women worldwide. However, the molecular mechanisms that lead to tumour development and progression remain poorly understood and there is a need to identify candidate genes associated with primary and metastatic breast cancer progression and prognosis. In this study, candidate genes associated with prognosis of primary and metastatic breast cancer were explored through a novel bioinformatics approach. Primary and metastatic breast cancer tissues and adjacent normal breast tissues were evaluated to identify biomarkers characteristic of primary and metastatic breast cancer. The Cancer Genome Atlas-breast invasive carcinoma (TCGA-BRCA) dataset (ID: HS-01619) was downloaded using the mRNASeq platform. Genevestigator 8.3.2 was used to analyse TCGA-BRCA gene expression profiles between the sample groups and identify the differentially-expressed genes (DEGs) in each group. For each group, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were used to determine the function of DEGs. Networks of protein–protein interactions were constructed to identify the top hub genes with the highest degree of interaction. Additionally, the top hub genes were validated based on overall survival and immunohistochemistry using The Human Protein Atlas. Of the top 20 hub genes identified, four (KRT14, KIT, RAD51, and TTK) were considered as prognostic risk factors based on overall survival. KRT14 and KIT expression levels were upregulated while those of RAD51 and TTK were downregulated in patients with breast cancer. The four proposed candidate hub genes might aid in further understanding the molecular changes that distinguish primary breast tumours from metastatic tumours as well as help in developing novel therapeutics. Furthermore, they may serve as effective prognostic risk markers based on the strong correlation between their expression and patient overall survival.

The ELEANOR noncoding RNA expression contributes to cancer dormancy and predicts late recurrence of estrogen receptor-positive breast cancer.

The recurrence risk of estrogen receptor (ER)‐positive breast cancer remains high for a long period of time, unlike other types of cancer. Late recurrence reflects the ability of cancer cells to remain dormant through various events, including cancer stemness acquisition, but the detailed mechanism is unknown. ESR1 locus enhancing and activating noncoding RNAs (ELEANORS) are a cluster of nuclear noncoding RNAs originally identified in a recurrent breast cancer cell model. Although their functions as chromatin regulators in vitro are well characterized, their roles in vivo remain elusive. In this study, we evaluated the clinicopathologic features of ELEANORS, using primary and corresponding metastatic breast cancer tissues. The ELEANOR expression was restricted to ER‐positive cases and well‐correlated with the ER and progesterone receptor expression levels, especially at the metastatic sites. ELEANORS were detected in both primary and metastatic tumors (32% and 29%, respectively), and frequently in postmenopausal cases. Interestingly, after surgery, patients with ELEANOR‐positive primary tumors showed increased relapse rates after, but not within, 5 years. Multivariate analysis showed that ELEANORS are an independent recurrence risk factor. Consistently, analyses with cell lines, mouse xenografts, and patient tissues revealed that ELEANORS upregulate a breast cancer stemness gene, CD44, and maintain the cancer stem cell population, which could facilitate tumor dormancy. Our findings highlight a new role of nuclear long noncoding RNAs and their clinical potential as predictive biomarkers and therapeutic targets for late recurrence of ER‐positive breast cancer.

Small nucleolar RNA SNORA71A promotes epithelial-mesenchymal transition by maintaining ROCK2 mRNA stability in breast cancer.

Metastasis is the primary reason of death in patients with cancer. Small nucleolar noncoding RNAs (snoRNAs) are conserved 60–300 nucleotide noncoding RNAs, involved in post‐transcriptional regulation of mRNAs and noncoding RNAs. Despite their essential roles in cancer, the roles of snoRNAs in epithelial‐mesenchymal transition (EMT)‐induced metastasis have not been studied extensively. Here, we used small RNA sequencing to screen for snoRNAs related to EMT and breast cancer metastasis. We found a higher expression of SNORA71A in metastatic breast cancer tissues compared to nonmetastatic samples. Additionally, SNORA71A promoted the proliferation, migration, invasion and EMT of MCF‐7 and MDA‐MB‐231 cells. Mechanistically, SNORA71A elevated mRNA and protein levels of ROCK2, a negative regulator of TGF‐β signaling. Rescue assays showed ROCK2 abrogated the SNORA71A‐mediated increase in proliferation, migration, invasion and EMT. Binding of SNORA71A to mRNA stability regulatory protein G3BP1, increased ROCK2 mRNA half‐life. Furthermore, G3BP1 depletion abolished the SNORA71A‐mediated upregulation of ROCK2. In vivo, SNORA71A overexpression promoted breast tumor growth, and SNORA71A knockdown inhibited breast cancer growth and metastasis. We suggest SNORA71A enhances metastasis of breast cancer by binding to G3BP1 and stabilizing ROCK2.

The relation between ACKR4 and CCR7 genes expression and breast cancer metastasis

AimsBreast cancer is the most severe malignant tumor in women. Chemokines and their receptors appear to be implicated in tumorigenesis and metastatic pattern. Also the scavenger atypical chemokine receptors are emerging as crucial regulators for the availability of chemokines. Therefore the aim of the present study is to evaluate the expression of CCR7, ACKR4 and their ligand; CCL21 in human breast cancer. Main methodsIn this study, RT-PCR was done to detect the expression of CCR7 and ACKR4 in 50 non-metastatic and 30 metastatic breast cancer tissue. Also CCL21 level in the serum of study group was detected by ELISA. The expression of all markers is compared to 80 control healthy individual. Key findingsOur results revealed the increase in expression of CCR7 and CCL21 level in metastatic group compared to non-metastatic and control groups while ACKR4 expression is significantly increased in breast tissues of non-metastatic patients compared to both control and metastatic groups. Also there was significant positive correlation between CCR7 expression and CCL21 level in cancer patients and significant negative correlation between ACKR4 and both CCR-7 and CCL21 in both non-metastatic and metastatic cancer groups. SignificanceThus, it might be elucidating that ACKR4 and CCR7 could be a novel target for tumor therapy as targeting the chemokine-receptors axis might represent a powerful tool to prevent infiltration and metastasis and consequently improve cancer prognosis and treatment.

Dysregulation of tumour microenvironment driven by circ-TPGS2/miR-7/TRAF6/NF-κB axis facilitates breast cancer cell motility

Tumour microenvironment (TME) is frequently remodelled and deregulated in cancer development and progression. The underlying mechanisms are complex, multifactorial and largely unknown. Circular RNA (circRNA) is an endogenous RNA with a covalently closed loop that plays critical roles in the pathological and physiological processes of the organism. Here, we identified a TME-associated circRNA, circ-TPGS2, which promoted breast cancer (BC) cell dissemination via altering TME. High circ-TPGS2 was observed in metastatic BC tissues and cell lines in comparison to respective normal controls, which was linked to poor overall and recurrence-free survival. Overexpression of circ-TPGS2 notably promoted cell migration, while silencing of circ-TPGS2 resulted in an opposite trend. Moreover, circ-TPGS2 increased pro-inflammatory chemokine production and evoked tumour-associated inflammation by recruiting neutrophils via autocrine and paracrine manners. In terms of mechanism, circ-TPGS2 acted as a sponge of miR-7 and elevated TRAF6, leading to p65 phosphorylation and nuclear translocation, ultimately activating NF-κB signalling. In addition, p65 was abundantly occupied on circ-TPGS2 promoter, activating circ-TPGS2 transcription, thus, forming a positive feedback loop that amplified the pro-metastasis effect of circ-TPGS2. Taken together, our data for the first time reveal the biological implication of circ-TPGS2 in BC, it triggers TME reshaping that facilitates BC metastasis through the miR-7/TRAF6/NF-κB signalling cascade.

Iodine Map Radiomics in Breast Cancer: Prediction of Metastatic Status.

Simple SummaryEarly and accurate diagnosis of breast cancer that has spread to other organs and tissues is crucial, as therapeutic decisions and outcome expectations might change. Computed tomography (CT) is often used to detect breast cancer’s spread, but this method has its weaknesses. The computer-assisted technique “radiomics” extracts grey-level patterns, so-called radiomic features, from medical images, which may reflect underlying biological processes. Our retrospective study therefore evaluated whether breast cancer spread can be predicted by radiomic features derived from iodine maps, an application on a new generation of CT scanners visualizing tissue blood flow. Based on 77 patients with newly diagnosed breast cancer, we found that this approach might indeed predict cancer spread to other organs/tissues. In the future, radiomics may serve as an additional tool for cancer detection and risk assessment.Dual-energy CT (DECT) iodine maps enable quantification of iodine concentrations as a marker for tissue vascularization. We investigated whether iodine map radiomic features derived from staging DECT enable prediction of breast cancer metastatic status, and whether textural differences exist between primary breast cancers and metastases. Seventy-seven treatment-naïve patients with biopsy-proven breast cancers were included retrospectively (41 non-metastatic, 36 metastatic). Radiomic features including first-, second-, and higher-order metrics as well as shape descriptors were extracted from volumes of interest on iodine maps. Following principal component analysis, a multilayer perceptron artificial neural network (MLP-NN) was used for classification (70% of cases for training, 30% validation). Histopathology served as reference standard. MLP-NN predicted metastatic status with AUCs of up to 0.94, and accuracies of up to 92.6 in the training and 82.6 in the validation datasets. The separation of primary tumor and metastatic tissue yielded AUCs of up to 0.87, with accuracies of up to 82.8 in the training, and 85.7 in the validation dataset. DECT iodine map-based radiomic signatures may therefore predict metastatic status in breast cancer patients. In addition, microstructural differences between primary and metastatic breast cancer tissue may be reflected by differences in DECT radiomic features.

A well plate-based multiplexed platform for incorporation of organoids into an organ-on-a-chip system with a perfusable vasculature.

Owing to their high spatiotemporal precision and adaptability to different host cells, organ-on-a-chip systems are showing great promise in drug discovery, developmental biology studies and disease modeling. However, many current micro-engineered biomimetic systems are limited in technological application because of culture media mixing that does not allow direct incorporation of techniques from stem cell biology, such as organoids. Here, we describe a detailed alternative method to cultivate millimeter-scale functional vascularized tissues on a biofabricated platform, termed 'integrated vasculature for assessing dynamic events', that enables facile incorporation of organoid technology. Utilizing the 3D stamping technique with a synthetic polymeric elastomer, a scaffold termed 'AngioTube' is generated with a central microchannel that has the mechanical stability to support a perfusable vascular system and the self-assembly of various parenchymal tissues. We demonstrate an increase in user familiarity and content analysis by situating the scaffold on a footprint of a 96-well plate. Uniquely, the platform can be used for facile connection of two or more tissue compartments in series through a common vasculature. Built-in micropores enable the studies of cell invasion involved in both angiogenesis and metastasis. We describe how this protocol can be applied to create both vascularized cardiac and hepatic tissues, metastatic breast cancer tissue and personalized pancreatic cancer tissue through incorporation of patient-derived organoids. Platform assembly to populating the scaffold with cells of interest into perfusable functional vascularized tissue will require 12-14 d and an additional 4 d if pre-polymer and master molds are needed.