Disease Progression In Breast Cancer Research Articles

Low social and family well-being is associated with greater RAGE ligand s100A8/A9 and interleukin-1 beta levels in metastatic breast cancer patients

Greater inflammatory signaling has been shown to promote breast cancer disease progression and poorer clinical outcomes. Lower social support and social well-being have been related to greater inflammatory signaling and poorer clinical outcomes in women with non-metastatic breast cancer, and this appears to be independent of depression. However, little is known about these associations in women with metastatic disease. s100A8/A9 and interleukin 1 beta (IL-1β) proteins are widely studied in breast cancer and are considered as biomarkers of cancer progression or as having a causal role in carcinogenesis and cancer progression and metastasis via inflammatory signaling. The aim of this study was to examine the associations between less social/family well-being (SWB) and S100A8/A9 and IL-1β levels in women diagnosed with metastatic breast cancer. Sixty women (Mean age 58.95 ​± ​1.49) with a diagnosis of metastatic breast cancer participated in the study. The Functional Assessment of Cancer Therapy (FACT) social and family well-being (SWB) subscale and the Hospital Anxiety Depression Scale (HADS) were administered to patients undergoing a first- or second-line endocrine or oral chemotherapy treatment and who were not experiencing brain metastasis or visceral crisis. Salivary s100A8/A9 and IL-1β levels were assessed at 5PM on two consecutive days and averaged. Multiple regression tested the independent contribution of SWB on s100 A8/A9 and IL-1b while controlling for depression. Lower levels of SWB were associated with greater S100A8/A9 (ß ​= ​−0.345, p ​= ​0.007) and IL-1β (ß ​= ​−0.286, p ​= ​0.027) levels and these associations remained significant after controlling for depression. This work provides new evidence for the role of decreased SWB and greater s100A8/A9 and IL-1b levels in patients diagnosed with metastatic breast cancer. Psychosocial interventions that promote social support and positive social interactions through interpersonal skills may help metastatic breast cancer patients to improve their SWB. This may have salutary effects on cancer-promoting processes, which could provide psychological and physical health benefits.

Abstract PO-148: The role of annexin A6 in triple-negative breast cancer metabolism and disease progression

Abstract Triple-negative breast cancer (TNBC), which accounts for up to 17% of all breast cancer cases in the Unites States, disproportionately affects premenopausal African American and Hispanic women. Here, we report the synergistic function of the tumor suppressor annexin-A6 (AnxA6) and Lapatinib-resistance (Lap-R) in the context of cellular bioenergetics. Metabolic profiling of TNBC cell lines show extensive diversity amongst basal-like (BSL) vs mesenchymal-like (MSL) molecular subtypes. Down regulation of AnxA6 in AnxA6- expressing and Lap-R cell lines attenuated mitochondrial respiration, glycolytic function, and cellular ATP production capacity, decreasing the overall metabolic plasticity of the cell. Additionally, AnxA6-depletion altered lipid metabolism by enhancing the mitochondrial uptake of cytosolic fatty acids for β-oxidation. NMR-based metabolomics revealed that AnxA6 depleted and/or Lap-R TNBC cells have a greater dependency on gluconeogenic precursors including glycine, alanine, lactic acid, and oxaloacetic acid as survival mechanisms. Taken together, this study proposes that altered expression of AnxA6 is accompanied by significant bioenergetic adaptations and hence provide novel insights into the failure of EGFR-targeted therapies as therapeutic options and disease progression in patients with triple-negative breast cancer. Citation Format: Stephen D. Williams, Sarrah E. Widatalla, Amos M. Sakwe. The role of annexin A6 in triple-negative breast cancer metabolism and disease progression [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-148.

Stimulated Raman scattering microscopy with spectral phasor analysis: applications in assessing drug-cell interactions.

Statins have displayed significant, although heterogeneous, anti-tumour activity in breast cancer disease progression and recurrence. They offer promise as a class of drugs, normally used for cardiovascular disease control, that could have a significant impact on the treatment of cancer. Understanding their mode of action and accurately assessing their efficacy on live cancer cells is an important and significant challenge. Stimulated Raman scattering (SRS) microscopy is a powerful, label-free imaging technique that can rapidly characterise the biochemical responses of live cell populations following drug treatment. Here, we demonstrate multi-wavelength SRS imaging together with spectral phasor analysis to characterise a panel of breast cancer cell lines (MCF-7, SK-BR-3 and MDA-MB-231 cells) treated with two clinically relevant statins, atorvastatin and rosuvastatin. Label-free SRS imaging within the high wavenumber region of the Raman spectrum (2800–3050 cm−1) revealed the lipid droplet distribution throughout populations of live breast cancer cells using biocompatible imaging conditions. A spectral phasor analysis of the hyperspectral dataset enables rapid differentiation of discrete cellular compartments based on their intrinsic SRS characteristics. Applying the spectral phasor method to studying statin treated cells identified a lipid accumulating phenotype in cell populations which displayed the lowest sensitivity to statin treatment, whilst a weaker lipid accumulating phenotype was associated with a potent reduction in cell viability. This study provides an insight into potential resistance mechanisms of specific cancer cells towards treatment with statins. Label-free SRS imaging provides a novel and innovative technique for phenotypic assessment of drug-induced effects across different cellular populations and enables effective analysis of drug–cell interactions at the subcellular scale.

Aberrant calcium signalling downstream of mutations in TP53 and the PI3K/AKT pathway genes promotes disease progression and therapy resistance in triple negative breast cancer.

Triple-negative breast cancer (TNBC) is characterized as an aggressive form of breast cancer (BC) associated with poor patient outcomes. For the majority of patients, there is a lack of approved targeted therapies. Therefore, chemotherapy remains a key treatment option for these patients, but significant issues around acquired resistance limit its efficacy. Thus, TNBC has an unmet need for new targeted personalized medicine approaches. Calcium (Ca2+) is a ubiquitous second messenger that is known to control a range of key cellular processes by mediating signalling transduction and gene transcription. Changes in Ca2+ through altered calcium channel expression or activity are known to promote tumorigenesis and treatment resistance in a range of cancers including BC. Emerging evidence shows that this is mediated by Ca2+ modulation, supporting the function of tumour suppressor genes (TSGs) and oncogenes. This review provides insight into the underlying alterations in calcium signalling and how it plays a key role in promoting disease progression and therapy resistance in TNBC which harbours mutations in tumour protein p53 (TP53) and the PI3K/AKT pathway.

Circulating Tumor DNA as a Predictive Marker of Recurrence for Patients With Stage II-III Breast Cancer Treated With Neoadjuvant Therapy.

BackgroundPatients with stage II to III breast cancer have a high recurrence rate. The early detection of recurrent breast cancer remains a major unmet need. Circulating tumor DNA (ctDNA) has been proven to be a marker of disease progression in metastatic breast cancer. We aimed to evaluate the prognostic value of ctDNA in the setting of neoadjuvant therapy (NAT).MethodsPlasma was sampled at the initial diagnosis (defined as before NAT) and after breast surgery and neoadjuvant therapy(defined as after NAT). We extracted ctDNA from the plasma and performed deep sequencing of a target gene panel. ctDNA positivity was marked by the detection of alterations, such as mutations and copy number variations.ResultsA total of 95 patients were enrolled in this study; 60 patients exhibited ctDNA positivity before NAT, and 31 patients exhibited ctDNA positivity after NAT. A pathologic complete response (pCR) was observed in 13 patients, including one ER(+)Her2(-) patient, six Her2(+) patients and six triple-negative breast cancer (TNBC) patients. Among the entire cohort, multivariate analysis showed that N3 classification and ctDNA positivity after NAT were independent risk factors that predicted recurrence (N3, hazard ratio (HR) 3.34, 95% confidence interval (CI) 1.26 – 8.87, p = 0.016; ctDNA, HR 4.29, 95% CI 2.06 – 8.92, p < 0.0001). The presence of ctDNA before NAT did not affect the rate of recurrence-free survival. For patients with Her2(+) or TNBC, patients who did not achieve pCR were associated with a trend of higher recurrence (p = 0.105). Advanced nodal status and ctDNA positivity after NAT were significant risk factors for recurrence (N2 – 3, HR 3.753, 95% CI 1.146 – 12.297, p = 0.029; ctDNA, HR 3.123, 95% CI 1.139 – 8.564, p = 0.027). Two patients who achieved pCR had ctDNA positivity after NAT; one TNBC patient had hepatic metastases six months after surgery, and one Her2(+) breast cancer patient had brain metastasis 13 months after surgery.ConclusionsThis study suggested that the presence of ctDNA after NAT is a robust marker for predicting relapse in stage II to III breast cancer patients.

Metabolomics-Microbiome Crosstalk in the Breast Cancer Microenvironment.

Breast cancer, the most frequent cancer diagnosed among females, is associated with a high mortality rate worldwide. Alterations in the microbiota have been linked with breast cancer development, suggesting the possibility of discovering disease biomarkers. Metabolomics has emerged as an advanced promising analytical approach for profiling metabolic features associated with breast cancer subtypes, disease progression, and response to treatment. The microenvironment compromises non-cancerous cells such as fibroblasts and influences cancer progression with apparent phenotypes. This review discusses the role of metabolomics in studying metabolic dysregulation in breast cancer caused by the effect of the tumor microenvironment on multiple cells such as immune cells, fibroblasts, adipocytes, etc. Breast tumor cells have a unique metabolic profile through the elevation of glycolysis and the tricarboxylic acid cycle metabolism. This metabolic profile is highly sensitive to microbiota activity in the breast tissue microenvironment. Metabolomics shows great potential as a tool for monitoring metabolic dysregulation in tissue and associating the findings with microbiome expression.

A Phase I Study of LSZ102, an Oral Selective Estrogen Receptor Degrader, with or without Ribociclib or Alpelisib, in Patients with Estrogen Receptor-Positive Breast Cancer.

Data are sparse for oral selective estrogen receptor (ER) degraders (SERD) in cancer treatment. The investigational oral SERD LSZ102 was assessed in monotherapy and combination use in a phase I study. A phase I, multicenter, open-label dose-escalation study (NCT02734615) of LSZ102 alone (arm A; n = 77) or with ribociclib (arm B; n = 78) or alpelisib (arm C; n = 43) in heavily pretreated adults with histologically confirmed ER-positive breast cancer and prior disease progression. Arm A received LSZ102 200-900 mg/day; arm B, LSZ102 200-600 mg/day plus ribociclib 300-600 mg/day; arm C, LSZ102 300-450 mg/day plus alpelisib 200-300 mg/day. Key outcomes were dose-limiting toxicities (DLT) in the first 28-day treatment cycle, adverse events (AE), laboratory parameters, pharmacokinetics, biopsy ER protein, and investigator-assessed clinical response (RECIST v1.1). The most common AEs were gastrointestinal. Treatment-related serious AEs occurred in 10% of participants (19/198), mostly in arm C [10/43 (23%)]. DLTs occurred in: arm A, 5% (4/77); arm B, 3% (2/78); and arm C, 19% (8/43). LSZ102 exposure was slightly greater than dose proportional. On-treatment biopsy ER reductions were observed, with a trend toward an LSZ102 dose response. Objective response rates (95% confidence interval) were: arm A, 1.3% (0.0-7.0); arm B, 16.9% (9.3-27.1); and arm C, 7.0% (1.5-19.1), and clinical benefit rates 7.8% (2.9-16.2), 35.1% (24.5-46.8), and 20.9% (10.0-36.0), respectively. LSZ102 was well tolerated alone and with ribociclib and had a manageable safety profile with alpelisib. Preliminary clinical activity was observed in combination use.

Utility of ferritin and inflammatory biomarkers in the diagnosis of different stages of breast cancer.

Objectives:To assess the utility of ferritin and inflammatory biomarkers in the diagnosis of stages of breast cancer.Methods:The study consisted of 4 groups of 20 women, including the healthy control. The patients of group 1 comprised of stages I and II, group 2: stage III and group 3: stage IV of the disease. High sensitive C-reactive protein (hsCRP) and hepcidin were estimated by enzyme linked immunosorbent assay and ferritin by chemiluminescence immunoassay. The study was carried out in 2018 at Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry-6, India.Results:The breast cancer disease progression correlated negatively with hemoglobin (Hb) (r= -0.6937, p<0.0001) and with ferritin levels, had a positive correlation (r=0.8444, p<0.0001). Ferritin negatively correlated with Hb among the subjects of the study (r= -0.6130, p<0.0001). Hepcidin correlated positively with hsCRP (r=0.998, p<0.0001). The ferritin/Hb ratio >3.9516 was used to identify the possibility of breast cancer utilizing the receiver operator curve values (area under curve [AUC]=0.997, sensitivity: 96.7, specificity:100). Ferritin values >103.4 were used to differentiate stage 4 from stage 3 of the disease (AUC: 0.893, sensitivity: 100, specificity: 85).Conclusion:Ferritin and ferritin/Hb are helpful in the differential diagnosis of stages of breast cancer.

A systems-based framework to computationally describe putative transcription factors and signaling pathways regulating glycan biosynthesis.

Glycosylation is a common posttranslational modification, and glycan biosynthesis is regulated by a set of glycogenes. The role of transcription factors (TFs) in regulating the glycogenes and related glycosylation pathways is largely unknown. In this work, we performed data mining of TF–glycogene relationships from the Cistrome Cancer database (DB), which integrates chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA-Seq data to constitute regulatory relationships. In total, we observed 22,654 potentially significant TF–glycogene relationships, which include interactions involving 526 unique TFs and 341 glycogenes that span 29 the Cancer Genome Atlas (TCGA) cancer types. Here, TF–glycogene interactions appeared in clusters or so-called communities, suggesting that changes in single TF expression during both health and disease may affect multiple carbohydrate structures. Upon applying the Fisher’s exact test along with glycogene pathway classification, we identified TFs that may specifically regulate the biosynthesis of individual glycan types. Integration with Reactome DB knowledge provided an avenue to relate cell-signaling pathways to TFs and cellular glycosylation state. Whereas analysis results are presented for all 29 cancer types, specific focus is placed on human luminal and basal breast cancer disease progression. Overall, the article presents a computational approach to describe TF–glycogene relationships, the starting point for experimental system-wide validation.

Heterotypic clustering of circulating tumor cells and circulating cancer-associated fibroblasts facilitates breast cancer metastasis.

Cancer-associated fibroblasts (CAFs) are recruited to the tumor microenvironment (TME) and are critical drivers of breast cancer (BC) malignancy. Circulating tumor cells (CTCs) travel through hematogenous routes to establish metastases. CTCs circulate both individually and, more rarely, in clusters with other cell types. Clusters of CTCs have higher metastatic potential than single CTCs. Previously, we identified circulating CAFs (cCAFs) in patients with BC and found that while healthy donors had no CTCs or cCAFs, both were present in most Stage IV patients. cCAFs circulate individually, as cCAF-cCAF homotypic clusters, and in heterotypic clusters with CTCs. In this study, we evaluate CTCs, cCAFs, and heterotypic cCAF-CTC clusters in patients with stage I-IV BC. We evaluate the association of heterotypic clusters with BC disease progression and metastasis in a spontaneous mouse model. Using previously established primary BC and CAF cell lines, we examine the metastatic propensity of heterotypic cCAF-CTC clusters in orthotopic and tail vein xenograft mouse models of BC. Using an in vitro clustering assay, we determine factors that may be involved in clustering between CAF and BC cells. We report that the dissemination of CTCs, cCAFs, and clusters is an early event in BC progression, and we find these clusters in all clinical stages of BC. Furthermore, cCAFs-CTC heterotypic clusters have a higher metastatic potential than homotypic CTC clusters in vivo. We also demonstrate that the adhesion and stemness marker CD44, found on a subset of CTCs and CAF cells, is involved in heterotypic clustering of these cells. We identify a novel subset of circulating tumor cell clusters that are enriched with stromal CAF cells in BC patient blood and preclinical mouse models of BC metastasis. Our data suggest that clustering of CTCs with cCAFs augments their metastatic potential and that CD44 might be an important mediator of heterotypic clustering of cCAFs and BC cells.

Selected Articles from This Issue

A prior cancer diagnosis is common among solid organ transplant candidates, and immunosuppressive medications administered to transplant recipients may increase recurrence risk. Engels and colleagues used data from the United States transplant registry and 13 cancer registries to assess cancer-specific survival among 10,524,326 cancer patients, 5425 of whom (0.05%) subsequently underwent transplantation. Transplantation was not associated with decreased cancer-specific survival for any cancer site. Protective associations were observed for patients with breast cancer, non-Hodgkin lymphoma, and myeloma. These results partly reflect careful transplant candidate selection and do not demonstrate a detrimental effect of immunosuppression on cancer-specific survival, supporting current management strategies.Chang and colleagues analyzed BOp. data across 3,460 adults in Wave 1 of the PATH Study, including current exclusive smokeless tobacco (ST) use among never smokers (primary ST users) and former smokers (secondary ST users), exclusive cigarette smokers, dual users of ST and cigarettes, former smokers, and never tobacco users. BOPH concentrations among primary ST users were similar to never tobacco users and former smokers. Dual users had significantly higher sICAM-1, IL-6, and F2-isoprostane compared to never tobacco users, but looked similar to exclusive smokers. ST use alone and in combination with smoking may affect levels of inflammatory and oxidative stress levels.Recruitment of stromal cells into the tumor microenvironment may influence disease progression and prognosis in breast cancer. By applying machine learning algorithms to digitized H&amp;E-stained sections for 2,084 breast cancer patients from China, Poland, and the United States, Abubakar and colleagues characterized tumor-associated stromal cellular density (SCD) as the percentage of tumor-stroma that is occupied by nucleated cells. The authors found increasing SCD to be independently predictive of poor clinical outcomes among patients with hormone receptor-positive breast cancer from all three study populations. The findings suggest that SCD assessment on H&amp;E-stained sections, which are routinely performed and widely available, could help refine prognostic assessment and treatment decision-making for patients with hormone receptor-positive breast cancer.Many women undergo risk-reducing oophorectomy (RRO) for ovarian cancer prevention at the time of hysterectomy. The abrupt decline in circulating hormones after RRO may impact body composition, particularly in premenopausal women. In this diverse national sample of women, Karia and colleagues observed higher total and regional (trunk, arms, legs) fat mass and lower lean mass among women with hysterectomy and RRO than those with hysterectomy alone or no surgery. Point estimates were stronger among women &lt;45 years at surgery and those with a normal BMI. Longitudinal studies are needed to assess changes in body composition after RRO and to test interventions to mitigate these effects.

Abstract 589: CCR5 upregulation in two subtypes of tumor associated circulating cells predict worse prognosis in metastatic breast cancer

Abstract Background: CCR5 is a motility chemokine receptor implicated in tumor progression, whose activation and endocytosis may identify highly aggressive breast cancer (BC) subtypes likely to spread via the circulatory system. We first studied the activation and endocytosis of CCR5 in response to its ligand CCL5 (RANTES) in the model BC cell line MDA-MB231. We then screened two types of circulating tumor-associated cells (TACs) with known negative clinical outcomes, 1) circulating tumor cells (CTCs) and 2) cancer-associated macrophage-like (CAMLs) cells, to evaluate CCR5 upregulation in relation to disease progression in metastatic breast cancer (mBC). Methods: MB231 cells were used to visualize CCR5 activation within the cell's surface and intracellular space using an anti-CCR5 antibody after stimulation with RANTES. MB231 cells were stained with LAMP-1A to verify co-localization of CCR5 and RANTES to endosomes. Anonymized peripheral blood samples from (n=54) mBC patients were obtained in accordance with local IRB regulations and with informed consent. TACs were isolated using a low-flow CellSieve Microfiltration system and stained for Cytokeratin, CD45, and CCR5. CCR5 expression signal and localization were evaluated on all TACs via fluorescence microscopy. Results: CCR5 in MB231 cells appeared as ~1μm clusters known as “CCR5 pools”, which can be found on the cell's surface, within the cytoplasm, and in the nuclear space. CCR5 pools were upregulated in the presence of RANTES, which appears to relate to endocytosis and nuclear translocation. Within the TACs of mBCs, we found similar patterns of CCR5 pooling with 70% of patients (n=38) having CCR5+ CAMLs and 41% (n=22) having CCR5+ CTCs. It was found that higher numbers of CCR5 pools (≥10pools/cell) were correlated to a 2-fold increase in both CTCs and CAMLs in circulation. In the analysis of outcome data, it was determined that patients having ≥10 CCR5 pools on their CAMLs was a highly significant predictor of worse progression free survival (PFS) and overall survival (OS) (PFS HR=4.8, CI 95% 1.9-11.6, p=0.0015) and (OS HR=3.6, CI 95% 1.3-10.0, p=0.0331). In CTCs ≥10 pools/cell was not a significant predictor for worse PFS and OS. However, ≥5 CCR5 pools/cell significantly predicted for worse PFS but not OS (PFS HR=2.9, CI 95% 1.2-7.4, p=0.0418 and OS HR=3.3, CI 95% 1.1-9.3, p= 0.0539). Conclusion: We observed the activation and endocytosis of CCR5 pools using RANTES in MD-MB231 cell lines, and then demonstrated that this upregulated motility pathway appears to be conserved in migratory cells in mBC patients. Further, this upregulation of CCR5 appears to correlate with worse clinical outcomes, which may provide an explanation for how tumor-associated cells move into circulation and acquire metastatic potential. Citation Format: Ashvathi Raghavakaimal, Massimo Cristofanilli, Cha-Mei Tang, R. K. Alpaugh, Kirby P. Gardener, Saranya Chumsri, Daniel L. Adams. CCR5 upregulation in two subtypes of tumor associated circulating cells predict worse prognosis in metastatic breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 589.

Regulation of Platelet-Derived ADAM17: A Biomarker Approach for Breast Cancer?

Tumor progression and metastasis are critically dependent on the tumor microenvironment. A disintegrin and metalloproteinase 17 (ADAM17) is associated with shedding of several substrates involved in tumor progression and known to be expressed by platelets of healthy donors and patients with solid tumors. Here, we report that platelet-derived ADAM17 (pADAM17) is regulated upon platelet activation of breast cancer patients, but not of healthy individuals. The observed downregulation of pADAM17 on platelets of cancer patients correlated with clinical parameters related to tumor progression including tumor stage and the occurrence of metastasis. Our data identify an association between platelet activation, modulation of platelet-derived ADAM17, and metastasis. In conclusion, we demonstrate that further development of pADAM17 as a liquid biomarker is warranted for monitoring disease progression in breast cancer.

Evolution of fibroblasts in the lung metastatic microenvironment is driven by stage-specific transcriptional plasticity.

Mortality from breast cancer is almost exclusively a result of tumor metastasis, and lungs are one of the main metastatic sites. Cancer-associated fibroblasts are prominent players in the microenvironment of breast cancer. However, their role in the metastatic niche is largely unknown. In this study, we profiled the transcriptional co-evolution of lung fibroblasts isolated from transgenic mice at defined stage-specific time points of metastases formation. Employing multiple knowledge-based platforms of data analysis provided powerful insights on functional and temporal regulation of the transcriptome of fibroblasts. We demonstrate that fibroblasts in lung metastases are transcriptionally dynamic and plastic, and reveal stage-specific gene signatures that imply functional tasks, including extracellular matrix remodeling, stress response, and shaping the inflammatory microenvironment. Furthermore, we identified Myc as a central regulator of fibroblast rewiring and found that stromal upregulation of Myc transcriptional networks is associated with disease progression in human breast cancer.

A comparative molecular dynamic simulation study on potent ligands targeting mTOR/FRB domain for breast cancer therapy.

Our study aimed to develop and find out the best drug candidate against the mechanistic target of rapamycin (mTOR/FRB) domain having a critical role in the aetiology of breast cancer. The FKBP12-rapamycin-binding (FRB) domain in the essential phosphoinositide 3 kinase/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway has been a vital player in the disease progression in breast cancer. By using structure-based drug designing , the best possible targets have been identified and developed. The three-dimensional structure of the target protein was generated using I-TASSER. The ligands were generated against the most suitable target active site using standard tools for active site identification. Furthermore, the seed molecule was drawn using Chemsketch, which was then grown into the pocket using Ligbuilder. The obtained ligands were further validated using online programs for bioavailability and toxicity, followed by molecular dynamic simulations. The study concludes that the equilibrated NVT-NPT complexes indicate LIG2 stability over LIG3. RMSD and RMSF have shown that the complex of LIG2 is more stable than LIG3. LIG2 has the potential antagonistic properties to target the mTOR/FRB domain and has therapeutic implications for breast cancer.

O80: GREMLIN-1 PROMOTES TUMOURIGENESIS AND METASTASIS IN HER2 POSITIVE BREAST CANCER

Abstract Introduction HER2 over-expression denotes poor prognosis in breast cancers.Bone morphogenetic protein(BMP) signalling is known to interact with EGF signalling, co-regulating breast cancer progression.BMP antagonist Gremlin-1 may influence breast cancer disease progression, but this remains unexplored in HER2 positive breast cancers. Method GREM1 and HER2 expression, and clinical outcomes were examined in clinical cohorts.GREM1 overexpression or pEF control plasmid were transduced into BT474 HER2+breast cancer cells. In vitro function tests using BT474 pEF and BT474GREM1cells include 2D/3D growth, migration, and expression of epithelial to mesenchymal transition(EMT)markers. Signalling cascades were examined in BT474 treated with RhGremlin-1. In vivo, BALB/c nude mice underwent either mammary injection or intra-cardiac injection of BT474pEF or BT474GREM1 cells and disease burden assessed. Result GREM1 expression correlates with HER2 in breast tumours(p=0.03) and is higher in metastatic HER2 positive cancers (p = 0.04). HER2 positive patients with high GREM1 have poor survival(p = 0.0002). BT474GREM1cells have up-regulated markers of EMT compared to control. BT474 RhGremlin-1 treated cells have active AKT pathway signalling, independent of BMP signalling. In vitro, BT474GREM1cells significantly proliferate and migrate compared to control(p&amp;lt;0.05 and p &amp;lt; 0.001).This is confirmed in vivo, BT474GREM1 mice grew significantly larger mammary tumours(p&amp;lt;0.05) and had more PETCT metastatic hotspots. Conclusion Gremlin-1 is correlated with poor outcomes in HER2 patients and promotes breast cancer cellular growth, migration and metastasis.Gremlin-1 is a novel area of research with potential as a prognostic biomarker and therapeutic target for personalised, effective, breast cancer outcomes. Take-home message BMP antagonists are gaining interest for their potential in breast cancer prognosis and therapeutics.This novel area of research shows BMP antagonist Gremlin-1 is of importance in HER2 positive breast cancers. DRAGONS DEN

Upregulation of LINC00511 expression by DNA hypomethylation promotes the progression of breast cancer

LINC00511 is a newly discovered long intergenic nonprotein-coding RNA (Ribonucleic acid) with unknown. Differential gene expression analysis was performed on breast cancer microarray data, and the upregulated expression of LINC00511 in breast cancer tissues and breast cancer cell lines was verified by qRT-PCR (quantitative Reverse Transcription-Polymerase Chain Reaction). A cohort study revealed a correlation between the expression of LINC00511 and the clinicopathological features in breast cancer patients. The effects of LINC00511 on breast cancer migration and invasion were studied in vitro. Then, an experiment using the Illumina Infinium Human Methylation450 Beadchip data was conducted to study the role of DNA (Deoxyribonucleic acid) methylation in LINC00511 expression, and DAVID (Database for Annotation, Visualization and Integrated Discovery) Functional Annotation Bioinformatics Microarray Analysis was used to determine the biological functions and potential pathways of LINC00511 in breast cancer. Then, LINC00511 and key genes associated with breast cancer disease progression were further studied in TCGA (The Cancer Genome Atlas), and western blotting was used to verify the results at the protein level. Finally, we further studied the effect of LINC00511 on Panobinostat drug sensitivity in breast cancer and its effect on the prognosis of breast cancer patients. LINC00511 was upregulated in breast cancer patients. The expression of LINC00511 was closely related to lymph node metastasis, tumor size and molecular subtypes of breast cancer. The in vitro studies revealed that LINC00511 could promote the migration and invasion in MDA-MB-231 and MCF-7 cells. In terms of mechanism, DNA hypomethylation promoted the expression of LINC00511, furthermore LINC00511 promoted the expression of Wnt10A, E2F2, TGFA, and MET, which participate in the progression of breast cancer. In addition, LINC00511 reduced the sensitivity of breast cancer cells to Panobinostat. Moreover, breast cancer patients with a high expression of LINC00511 had a poor prognosis. DNA hypomethylation promotes the expression of LINC00511 in breast cancer, and LINC00511 promotes the progression of breast cancer by upregulating Wnt10A, E2F2, TGFA and MET. High expression of LINC00511 is associated with poor prognosis. Our study identified the mechanism of LINC00511 upregulation and provides novel information on the progression of breast cancer.

Prolonged Time from Diagnosis to Breast-Conserving Surgery is Associated with Upstaging in Hormone Receptor-Positive Invasive Ductal Breast Carcinoma

BackgroundTime to surgery (TTS) has been suggested to have an association with mortality in early-stage breast cancer.ObjectiveThis study aims to determine the association between TTS and preoperative disease progression in tumor size or nodal status among women diagnosed with clinical T1N0M0 ductal breast cancer.MethodsWomen diagnosed with clinical T1N0M0 ductal breast cancer who had breast-conserving surgery as their first definitive treatment between 2010 and 2016 (n = 90,405) were analyzed using the National Cancer Database. Separate multivariable logistic regression models for hormone receptor (HR)-positive and HR-negative patients, adjusted for clinical and demographic variables, were used to assess the relationship between TTS and upstaging of tumor size (T-upstaging) or nodal status (N-upstaging).ResultsT-upstaging occurred in 6.76% of HR-positive patients and 11.00% of HR-negative patients, while N-upstaging occurred in 12.69% and 10.75% of HR-positive and HR-negative patients, respectively. Among HR-positive patients, odds of T-upstaging were higher for 61–90 days TTS (odds ratio [OR] 1.18, 95% confidence interval [CI] 1.05–1.34) and ≥91 days TTS (OR 1.47, 95% CI 1.17–1.84) compared with ≤30 days TTS, and odds of N- upstaging were higher for ≥91 days TTS (OR 1.35, 95% CI 1.13–1.62). No association between TTS and either T- or N-upstaging was found among HR-negative patients. Other clinical and demographic variables, including grade, tumor location, and race/ethnicity, were associated with both T- and N-upstaging.ConclusionTTS ≥61 and ≥91 days was a significant predictor of T- and N-upstaging, respectively, in HR-positive patients; however, TTS was not associated with upstaging in HR-negative breast cancer. Delays in surgery may contribute to measurable disease progression in T1N0M0 ductal breast cancer.

Abstract PS17-54: Hepatocyte growth factor regulates the expression of chemokine family in vascular endothelial cells; potential implications in clinical breast cancer

Abstract Background. Hepatocyte growth factor (HGF) is a powerful cellular migration stimulating cytokine (a motogen) of both epithelial and endothelial cells. The induced cell motility is mainly in the form of chemokinesis, namely undirectional movement of the cells in response to exogenous stimuli. HGF is a key angiogenic and lymphangiogenic factor, partially through its effects on endothelial cellular migration, adhesiveness and morphogenesis, which in turn contribute to the growth and spread of cancer cells including breast cancer cells. It has been suggested that HGF might directly or indirectly mediate chemotactic cell movement, namely chemotaxis. Here, we investigated the impact of HGF on the expression of the chemokine family, including the CCL (C-C Motif Chemokine Ligands) family and the CXCL (C-X-C Motif Chemokine Ligands) family and the subsequent consequence of these chemokines on endothelial and cancer cells. Method. Human recombinant hepatocyte growth factor and human recombinant CCLs were used in the study. Human vascular endothelial cells were challenged with HGF and their gene expression was profiled using gene microarray technology. The effects of the responsive chemokines were further tested on endothelial cells and the expression of the respective CCLs was knocked down in endothelial cells by way of siRNA. The impact on the paracellular permeability was assessed by PCP (paracellular permeability) assay using a fluorescence tracer and by transendothelial electrical resistance (TER). TER and cellular migration were also evaluated using automated electric cell substrate impedance sensing (ECIS) methods. Results. Within the concentrations known to induce biological functions in endothelial cells, HGF had a profound upregulatory effect on the expression of multiple chemokines, including CCL14, CCL20, CCL21, CCL28. CXCL family members also responded to HGF, including CXCL8 (p&amp;lt;0.001), CXCL3 (p&amp;lt;0.01), CXCL2 (p=0.01) and CXCL17 (p&amp;lt;0.05). We chose to further investigate the effects of a panel of CCLs which have significant clinical connections in breast cancer, notably CCL20 (otherwise known as LARC, Liver And Activation-Regulated Chemokine). High level CCL20 expression in breast cancer is correlated with a favourable relapse free survival (p=0.0021) of the patients (TCGA database, kmplot.com). CCL20 had a marginal inhibitory effect on the permeability of paracellular space, however, it had profound effects on the migration of cancer cells, seen by a marked increase in cellular migration. A similar stimulatory effect on cancer cells was seen in conditioned media from HGF activated endothelial cells, and partially abolished by siRNA to CCL20. Discussion. HGF exerts an upregulatory effect on the expression of certain chemokines from endothelial cells, which in turn acted on cancer cells by inducing chemotaxic effects. HGF, via regulation of chemokines, may regulate both directional and unidirectional migration of cancer cells, and impact the disease progression in breast cancer. Citation Format: Wenjing Gong, Andrew James Sanders, Tracey Amanda Martin, Yanan Gu, Ping Sun, Wen Guo Jiang. Hepatocyte growth factor regulates the expression of chemokine family in vascular endothelial cells; potential implications in clinical breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-54.

Abstract PS4-44: Expression of the claudin transmembrane tight junction protein family (CLDN) and the prediction value of a claudin subset to the clinical outcome of patients with breast cancer

Abstract Background: Tight Junction (TJ) proteins are directly or indirectly involved in breast cancer progression and metastasis where the mutual adhesiveness of cancer cells is significantly weaker than normal cells and so reduced cell-cell interaction results in a loss of normal tissue architecture (1). This adhesiveness is in addition to the role of the proteins in the control of the barrier functions of epithelial and endothelial cells. The claudin family (CLDN) of transmembrane proteins, the largest protein family of the tight junctions, are located in the TJ of cells of epithelial and endothelial origin where they are key in the maintenance of TJ function and have been increasingly shown to have important roles during key steps in the progression of cancer. We have previously demonstrated that the expression of some TJ molecules (occludin, claudins -5, -16, -20) are associated with disease progression in breast cancer (2-5). This study sought to evaluate the possible clinical and prognostic value of the entire CLDN family, in patients with breast cancer. Methods: Members of the claudin family of transmembrane TJ proteins were assessed and correlated with clinical and pathological parameters at the messenger level in a Cardiff breast cancer cohort. Breast cancer primary tumours (n=114) and matched background tissue (n=30) were processed for RNA extraction. RNA was reverse transcribed and quantified before analysis by Q-PCR CLDN-1 to -24. The expression profile was analysed against the clinical and pathological information and most importantly the clinical outcome of the patients. Results. Mammary tissues expressed varying levels of CLDNs. However, the levels of CLDN1, CLDN3, CLDN4, CLDN11, CLDN19, and CLDN22 were found to be significantly aberrant in breast cancer tissues compared with normal tissues. Members of the family, namely CLDN10, CLDN11, and CLDN18 were found to have a significant predictive value to the prognosis when using the Nottingham Prognostic Index. Of the claudin family, we identified eight members whose expression was negatively correlated with long term survival, and three members positively correlated with the survival. A comprehensive bioinformatic analyses of these CLDN members revealed a highly significant signature in predicting the clinical outcome. A favourable CLDN signature predicted a subset of the patients who all survived the ten-year followup, compared with those with unfavourable signature (69.1%), p&amp;lt;0.0001 (Log ranked). Likewise, the CLDN signature also successfully predicted the occurrence of breast cancer related incidence (97.6% vs 61.8%, p&amp;lt;0.0001). Multivariate analyses confirmed that the CLDN signature has a highly significant value as an independent predictor for both overall survival (p=0.001) and disease-free survival (p=0.001). Moreover, the predictive power was more profound for patients with ER negative and Her2 negative tumours. Discussion: Claudins, key tight junctional transmembrane proteins, have important prognostic value in human breast cancer. Together with their pivotal roles in controlling the adhesiveness and permeability of the cell layers, this family is a key player in the development and progression of breast cancer.