Phenotype Of Breast Cancer Cells Research Articles

Selection of RNA aptamers targeting hypoxia in cancer.

Hypoxia plays a crucial role in tumorigenesis and drug resistance, and it is recognised as a major factor affecting patient clinical outcome. Therefore, the detection of hypoxic areas within the tumour micro-environment represents a useful way to monitor tumour growth and patients’ responses to treatments, properly guiding the choice of the most suitable therapy. To date, non-invasive hypoxia imaging probes have been identified, but their applicability in vivo is strongly limited due to an inadequate resistance to the low oxygen concentration and the acidic pH of the tumour micro-environment. In this regard, nucleic acid aptamers represent very powerful tools thanks to their peculiar features, including high stability to harsh conditions and a small size, resulting in easy and efficient tumour penetration. Here, we describe a modified cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach that allows the isolation of specific RNA aptamers for the detection of the hypoxic phenotype in breast cancer (BC) cells. We demonstrated the effectiveness of the proposed method in isolating highly stable aptamers with an improved and specific binding to hypoxic cells. To our knowledge, this is the first example of a cell-SELEX approach properly designed and modified to select RNA aptamers against hypoxia-related epitopes expressed on tumour cell surfaces. The selected aptamers may provide new effective tools for targeting hypoxic areas within the tumour with great clinical potential.

KIF17 maintains the epithelial phenotype of breast cancer cells and curbs tumour metastasis

Kinesin superfamily motor protein 17 (KIF17) was previously identified downregulated in breast cancer and correlated with patient prognosis. However, its pathophysiological role in tumours remains unknown. Here, we confirmed that KIF17 was significantly under-expressed in breast cancer tissues and low KIF17 expression correlated with poor outcomes in patients with breast cancer. In vitro and in vivo experiments demonstrated that KIF17 overexpression in breast cancer cell lines significantly inhibited breast cancer invasion and metastasis. By establishing the lung metastatic MDA-MB-231 cell lines, we found a transient silence of KIF17 during the initiation of breast cancer metastasis. Further experiments revealed that KIF17 might suppress metastasis by regulating the level of acetylated tubulin to maintain cytoskeleton stability. Eventually, we found that the low expression of KIF17 in breast cancer is regulated by DNMT1-mediated 5-mC DNA methylation and epigenetic silencing. Decitabine can effectively improve the expression level of KIF17 in breast cancer cells. Our study demonstrates that KIF17 mediates microtubule acetylation to maintain the stability of microtubules, thereby inhibiting tumour invasion and metastasis.

MiR-302 Suppresses the Proliferation, Migration, and Invasion of Breast Cancer Cells by Downregulating ATAD2.

Simple SummaryATPase family AAA domain-containing protein 2 (ATAD2) overexpression is associated with poor survival and disease recurrence in multiple cancers. The current study aimed to investigate the expression and function of ATAD2 in breast cancer. Our results showed that ATAD2 expression was upregulated in human breast cancer tissues and cell lines, while ATAD2 knockdown inhibited the proliferation, migration, and invasion of breast cancer cells. Moreover, we provide evidence suggesting that miR-302 directly targets ATAD2 and thus modulates cancer cell proliferation, migration, and invasion in vitro. Moreover, ATAD2 overexpression rescued the inhibition of tumor growth caused by miR-302 in xenograft mice. These findings indicate that miR-302 plays a crucial role in inhibiting the malignant phenotypes of breast cancer cells by targeting ATAD2.Breast cancer is the most common malignant tumor in women. The ATPase family AAA domain-containing protein 2 (ATAD2) contains an ATPase domain and a bromodomain, and is abnormally expressed in various human cancers, including breast cancer. However, the molecular mechanisms underlying the regulation of ATAD2 expression in breast cancer remain unclear. This study aimed to investigate the expression and function of ATAD2 in breast cancer. We found that ATAD2 was highly expressed in human breast cancer tissues and cell lines. ATAD2 depletion via RNA interference inhibited the proliferation, migration, and invasive ability of the SKBR3 and T47D breast cancer cell lines. Furthermore, Western blot analysis and luciferase assay results revealed that ATAD2 is a putative target of miR-302. Transfection with miR-302 mimics markedly reduced cell migration and invasion. These inhibitory effects of miR-302 were restored by ATAD2 overexpression. Moreover, miR-302 overexpression in SKBR3 and T47D cells suppressed tumor growth in the xenograft mouse model. However, ATAD2 overexpression rescued the decreased tumor growth seen after miR-302 overexpression. Our findings indicate that miR-302 plays a prominent role in inhibiting the cancer cell behavior associated with tumor progression by targeting ATAD2, and could thus be a valuable target for breast cancer therapy.

GASP1 enhances malignant phenotypes of breast cancer cells and decreases their response to paclitaxel by forming a vicious cycle with IGF1/IGF1R signaling pathway

There is a potential correlation between G-protein-coupled receptor-associated sorting protein 1 (GASP1) and breast tumorigenesis. However, its biological function and underlying molecular mechanism in breast cancer have not been clearly delineated. Here, we demonstrated that GASP1 was highly expressed in breast cancers, and patients harboring altered GASP1 showed a worse prognosis than those with wild-type GASP1. Functional studies showed that GASP1 knockout significantly suppressed malignant properties of breast cancer cells, such as inhibition of cell proliferation, colony formation, migration, invasion and xenograft tumor growth in nude mice as well as induction of G1-phase cell cycle arrest, and vice versa. Mechanistically, GASP1 inhibited proteasomal degradation of insulin-like growth factor 1 receptor (IGF1R) by competitively binding to IGF1R with ubiquitin E3 ligase MDM2, thereby activating its downstream signaling pathways such as NF-κB, PI3K/AKT, and MAPK/ERK pathways given their critical roles in breast tumorigenesis and progression. IGF1, in turn, stimulated GASP1 expression by activating the PI3K/AKT pathway, forming a vicious cycle propelling the malignant progression of breast cancer. Besides, we found that GASP1 knockout obviously improved the response of breast cancer cells to paclitaxel. Collectively, this study demonstrates that GASP1 enhances malignant behaviors of breast cancer cells and decreases their cellular response to paclitaxel by interacting with and stabilizing IGF1R, and suggests that it may serve as a valuable prognostic factor and potential therapeutic target in breast cancer.

CYP1B1 Augments the Mesenchymal, Claudin-Low, and Chemoresistant Phenotypes of Triple-Negative Breast Cancer Cells.

Cytochrome P4501B1 (CYP1B1) is elevated in breast cancer. Studies indicate a relationship between CYP1B1 and aggressive cancer phenotypes. Here, we report on in vitro studies in triple-negative breast cancer cell lines, where knockdown (KD) of CYP1B1 was used to determine the influence of its expression on invasive cell phenotypes. CYP1B1 KD in MDA-MB-231 cells resulted in the loss of mesenchymal morphology, altered expression of epithelial–mesenchymal genes, and increased claudin (CLDN) RNA and protein. CYP1B1 KD cells had increased cell-to-cell contact and paracellular barrier function, a reduced rate of cell proliferation, abrogation of migratory and invasive activity, and diminished spheroid formation. Analysis of clinical breast cancer tumor samples revealed an association between tumors exhibiting higher CYP1B1 RNA levels and diminished overall and disease-free survival. Tumor expression of CYP1B1 was inversely associated with CLDN7 expression, and CYP1B1HI/CLDN7LOW identified patients with lower median survival. Cells with CYP1B1 KD had an enhanced chemosensitivity to paclitaxel, 5-fluorouracil, and cisplatin. Our findings that CYP1B1 KD can increase chemosensitivity points to therapeutic targeting of this enzyme. CYP1B1 inhibitors in combination with chemotherapeutic drugs may provide a novel targeted and effective approach to adjuvant or neoadjuvant therapy against certain forms of highly metastatic breast cancer.

ANKHD1 contributes to the malignant phenotype of triple-negative breast cancer cells.

Ankyrin repeat and KH domain-containing protein 1, ANKHD1, has been identified as a regulator of signaling pathways and cellular processes of relevance in carcinogenesis. However, the role of ANKHD1 in breast cancer remains unclear. The aim of the present study was to characterize the expression pattern and involvement of ANKHD1 in the malignant phenotype of breast cancer cell lines and to investigate the clinical relevance of ANKHD1 in a breast cancer context. Gene and protein expressions were assessed in the cell lines by quantitative reverse transcription PCR and Western blot analysis, respectively, and ANKHD1 silencing through siRNA transfection was conducted for further in vitro functional assays. The expression of ANKHD1 was identified in non-tumorigenic breast epithelium and breast cancer cell lines, but differences in cellular localization were found among the neoplasia subtypes. ANKHD1 silencing reduced the viability, clonogenicity, and migration of triple-negative breast cancer (TNBC) cells. Bioinformatics analyses demonstrated that patients with triple-negative basal-like 2 and mesenchymal breast cancer subtypes had high ANKHD1 expression associated with poor recurrence-free survival. Therefore, these data indicate that ANKHD1 relevance in breast cancer varies among its subtypes, indicating the importance of ANKHD1 in TNBC.

Abstract 1000: E-cadherin and EGFR interactions result in hyper-proliferation via ERK signaling in breast cancer

Abstract The loss of the intercellular adhesion molecule E-cadherin is a hallmark of the epithelial-mesenchymal transition (EMT), during which tumor cells transition into an invasive phenotype. Thus, E-cadherin has long been considered a tumor suppressor gene. However, recent studies have provided evidence that E-cadherin may promote metastasis rather than suppress it, suggesting oncogenic behavior. Here we provide data that E-cadherin plays an oncogenic role in breast cancer by promoting a hyper-proliferative phenotype in breast cancer cells via interaction with EGFR. This interaction results in the activation of the MEK/ERK signaling pathway, leading directly to changes in proliferation via transcription factors such as c-Fos. Pharmacological inhibition of MEK in E-cadherin positive breast cancer cells significantly decreases both tumor growth and macro-metastasis in vivo. This work provides evidence for a novel role of E-cadherin in breast tumor progression and identifies a potential new target to treat hyper-proliferative E-cadherin-positive breast tumors. Citation Format: Gabriella C. Russo, Ashleigh Crawford, David J. Clark, Julie Cui, Ryan Carney, Michelle N. Karl, Boyang Su, Batrholomew Starich, Tung-Shing Lee, Qiming Zhang, Pei-hsun Wu, Meng-Horng Lee, Hon S. Leong, Vito Rebecca, Hui Zhang, Denis Wirtz. E-cadherin and EGFR interactions result in hyper-proliferation via ERK signaling in 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 1000.

Abstract 3200: Examining the role of obesity and leptin signaling in triple negative breast cancer

Abstract Individuals with a high body mass index (BMI) have an increased risk of developing many adult cancers, including breast cancer. In triple negative breast cancer (TNBC), a clinically aggressive subtype of breast cancer, increased BMI is associated with more aggressive tumor types and a higher risk of recurrence. The differences in outcomes between obese and non-obese breast cancer patients is a consequence of the complex interplay between social, environmental, and physiological factors that contribute to the etiology of breast cancer. Therefore, understanding the complex signaling events in the obese tumor microenvironment is essential. Adipose stem cells (ASCs) are a component of the breast microenvironment, and are a subset of mesenchymal stem cells. ASCs from obese patients (obASCs) secrete higher levels various cytokines and adipokines that induce a more invasive phenotype in triple negative breast cancer cells compared to ASCs from lean individuals. Leptin, an adipokine that is expressed proportionally to fat mass, has been implicated in many cancers. Increased leptin and leptin receptor expression is associated with worse prognosis. This study seeks to examine the role of leptin signaling in triple negative breast cancer. Previous work in conjunction with a collaborating lab has shown that leptin signaling promotes metastasis and increased expression of epithelial-mesenchymal transition (EMT) markers in triple negative breast cancer. This project expands upon this work through using both patient-derived cell lines and and patient-derived xenografts (PDX), and examines the role of leptin signaling both in vitro and in vivo. To determine the effects of obesity upon a PDX model, a high fat diet was used to induce obesity in vivo. Exposure to conditioned media harvested from obASCs increased the percentage of TNBC cells that expressed cancer stem cell markers, whereas exposure to a small peptide antagonist of the leptin receptor decreased the percentage of cells with CSC markers. PDX tumors implanted into diet-induced obesity mice had an increased volume compared to tumors implanted into lean controls. Conclusions: These molecular differences may contribute to the differences in cancer outcomes between obese and lean individuals with breast cancer, and further study of the crosstalk between obASCs and TNBC is critical. Citation Format: Courtney Brock, Maryl Wright, Khoa Nguyen, Katherine Hebert, Madlin Alzoubi, Thomas Cheng, Bridgette Collins-Burow, Matthew Burow. Examining the role of obesity and leptin signaling in triple negative 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 3200.

Abstract 1523: Beta-2 adrenergic receptor and mu opioid receptor interact to increase the aggressiveness in triple negative breast cancer cells through activation of GSK3 signaling

Abstract Breast cancer is the world’s leading cause of cancer mortality in women worldwide. It has been shown that body stress hormones, such as norepinephrine, and opioid peptides interact to control the growth and progression of this cancer. Opioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control cellular functions in various cell systems. However, the underlying cell signaling mechanism remains unclear. To determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer (TNBC) cell lines MDA-MB-231 and MDA-MB-468, CRISPR, chemical inhibition or activation and, PCR Array technology to study aggressive tumor phenotype and signaling cascades. We found that knock down of the beta-adrenergic (B2AR) or mu-opioidergic receptor (MOR) system via the CRISPR technique or pharmacological agents induces a less aggressive phenotype in triple negative breast cancer cells both in vitro and in vivo, with the effect being potentiated when both receptor systems were inhibited together. The mRNA expression study using a PCR Array targeting Cancer Stem Cell genes shows that B2AR or MOR activated the glycogen synthase kinase 3 (GSK3) signaling cascade. The GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as was done by the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation. These data identify a role of B2AR and MOR interaction in the control of breast cancer cell growth and establish a possible role of the GSK3 signaling system in mediation of these two receptors’ cross talk. (Supported by NIH grant R01 CA20863201). Citation Format: Bénédicte Rousseau, Sengottuvelan Murugan, Ajay Palagani, Dipak K. Sarkar. Beta-2 adrenergic receptor and mu opioid receptor interact to increase the aggressiveness in triple negative breast cancer cells through activation of GSK3 signaling [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 1523.

Abstract 3135: Postmenopausal obese mammary adipocytes promote breast tumorigenesis via the LCN2/IL-6/STAT3 signaling axis

Abstract Obesity is associated with a substantially increased risk (~50%) and a poor prognosis of breast cancer (BC) in postmenopausal (PM) women. The mechanism(s) underlying obesity-related BC are not clearly understood and, to date, most studies focus on the systemic effects of subcutaneous or visceral adipocytes on BC. We hypothesized that the increased local presence of ‘obese’ mammary adipocytes within the breast microenvironment promotes the acquisition of an invasive and vascular breast tumor cell phenotype and thereby markedly accelerates tumor proliferation and progression. We first asked whether local interactions between mammary adipocytes and BC cells might promote tumor growth and if so, what the underlying mechanism(s) might be. BC cells (ER- and ER+), when treated with the secretome of mammary adipocytes from obese women (ObAdCM), upregulated potent angiogenic/inflammatory factors, including VEGF, bFGF, lipocalin-2 (LCN2), IL-6 and significantly suppressed the angiogenesis inhibitor Tsp-1. ObAdCM stimulated endothelial cell (EC) recruitment by BC cells and increased EC proliferation, indispensable steps in tumor neovascularization. PM ObAdCM significantly stimulated BC cell proliferation, migration and invasion. Both IL-6 and LCN2-neutralization resulted in significant reduction of the ObAdCM-stimulated BC cell migration and proliferation, suggesting that these factors play a role in obesity-mediated BC progression. ObAdCM induced pSTAT3/pAkt signaling in BC cells and IL-6 and LCN2 could independently stimulate pSTAT3 in BC cells. STAT3 inhibition abrogated the ObACDM-mediated effects on BC cell proliferation and migration, suggesting that the pro-tumorigenic effects of PM obese mammary adipocytes are mediated by LCN2/IL-6/STAT3 signaling. Comparative RNAseq analyses identified >30 differentially expressed genes (DEGs) in PM compared to pre-menopausal (Pre-M) obese mammary adipocytes. Furthermore, GO and KEGG enrichment analysis of DEGs suggested an upregulation of PI3K/Akt signaling, ECM-receptor interactions, focal adhesions and lipid/fatty acid metabolism in PM mammary adipocytes. Taken together, our results indicate that obese mammary adipocytes in the breast tumor microenvironment can promote migration, proliferation, invasion and the vascular phenotype of BC cells and suggest that targeting the LCN2/IL-6/STAT3 signaling axis may be a useful strategy in the treatment of obesity-driven breast tumorigenesis. [Supported by NIH RO1CA185530, the Breast Cancer Research Foundation, the Karp Family Foundation and the Nile Albright Research Foundation] Citation Format: Roopali Roy, Emily Man, Katherine Gonzalez, Lauren Merritt, Cassandra Daisy, Rama Aldakhallah, Liang Sun, Biju Isaac, Shira Rockowitz, Margaret Lotz-Bousvaros, Susan Pories, Marsha A. Moses. Postmenopausal obese mammary adipocytes promote breast tumorigenesis via the LCN2/IL-6/STAT3 signaling axis [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 3135.

Low expression of PCK2 in breast tumors contributes to better prognosis by inducing senescence of cancer cells.

Cell cycle arrest, one of the main characteristics of cellular senescence, has been described as a crucial barrier that needs to be bypassed for cancer progression. Typically, cellular senescence can be induced by multiple stresses including telomere shortening, oncogenic activation as well as therapy treatment, and contributes to the inhibition of epithelial-mesenchymal transition (EMT), tumor suppression or progression depending on the senescence-associated secretory phenotype (SASP) components. However, the mechanisms underlying cancer cell senescence remain partially understood. Here, according to METABRIC database, we identified that patients with senescent-like breast tumors show better short-term survival, lower tendency of neoplasm histological grades, lower tumor stages, and negative status of estrogen receptor (ER) and progesterone receptor (PR) compared with non-senescent ones. Interestingly, Kyoto encyclopedia of genes and genomes (KEGG) analysis identified insulin signaling was significantly repressed in senescent breast tumors. Further verification in cultured breast cancer cells indicated that phosphoenolpyruvate carboxykinase 2 (PCK2) was significantly inhibited after therapy treatment. In addition, knockdown of PCK2 induced a senescent phenotype of breast cancer cells. Moreover, comparing with the non-senescent group, the senescent breast cancers displayed lower EMT capacity both in patients and breast cancer cell lines after knocking down PCK2. In conclusion, we described for the first time that low expression level of PCK2 may contribute to better prognosis via triggering senescent phenotype and thereby inhibiting EMT capacity in breast cancers.

NSrp70 suppresses metastasis in triple-negative breast cancer by modulating Numb/TβR1/EMT axis.

Alternative splicing of mRNA precursors allows cancer cells to create different protein isoforms that promote growth and survival. Compared to normal cells, cancer cells frequently exhibit a higher diversity of their transcriptomes. A comprehensive understanding of splicing regulation is required to correct the splicing alterations for the future precision oncology. A quantitative proteomic screen was performed to identify the regulators associated the metastasis in triple-negative breast cancer. Multiple in vitro and in vivo functional analyses were used to study the effects of NSrp70 on breast cancer metastasis. Next, transcriptomic sequencing (RNA-seq) and alternative splicing bioinformatics analysis was applied to screen the potential targets of NSrp70. Moreover, in vitro splicing assays, RNA pull-down, and RNA immunoprecipitation assay were used to confirm the specific binding between NSrp70 and downstream target genes. Furthermore, the prognostic value of NSrp70 was analyzed in a cohort of patients by performing IHC. We uncovered NSrp70 as a novel suppressor of breast cancer metastasis. We discovered that NSrp70 inhibited the skipped exon alternative splicing of NUMB, promoted the degradation of transforming growth factor receptor 1 through lysosome pathway, and regulated TGFβ/SMAD-mediated epithelial-mesenchymal transition phenotype in breast cancer cells. Furthermore, high NSrp70 expression correlated with a better prognosis in breast cancer patients. Our findings revealed that splicing regulator NSrp70 serves as a metastasis suppressor.

Beta 2 adrenergic receptor and mu opioid receptor interact to potentiate the aggressiveness of human breast cancer cell by activating the glycogen synthase kinase 3 signaling

BackgroundOpioid and beta-adrenergic receptors are recently shown to cross talk via formation of receptor heterodimers to control the growth and proliferation of breast cancer cells. However, the underlying cell signaling mechanism remained unclear.MethodsTo determine the effect of the interaction of the two systems in breast cancer, we employed triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468, CRISPR or chemical inhibition or activation of beta-adrenergic receptors (B2AR) and mu-opioid receptors (MOR) gene, and PCR array technology and studied aggressive tumor phenotype and signaling cascades.ResultsWe show here that in triple-negative breast cancer cells, the reduction in expression B2AR and MOR by genetic and pharmacological tools leads to a less aggressive phenotype of triple-negative breast cancer cells in vitro and in animal xenografts. Genomic analysis indicates the glycogen synthase kinase 3 (GSK3) pathway as a possible candidate messenger system involved in B2AR and MOR cross talk. GSK3 inactivation in MDA-MB-231 and MDA-MB-468 cells induced similar phenotypic changes as the inhibition of B2AR and/or MOR, while a GSK3 activation by wortmannin reversed the effects of B2AR and/or MOR knockdown on these cells. GSK3 inactivation also prevents B2AR agonist norepinephrine or MOR agonist DAMGO from affecting MDA-MB-231 and MDA-MB-468 cell proliferation.ConclusionsThese data confirm a role of B2AR and MOR interaction in the control of breast cancer cell growth and identify a possible role of the GSK3 signaling system in mediation of these two receptors’ cross talk. Screening for ligands targeting B2AR and MOR interaction and/or the GSK3 system may help to identify novel drugs for the prevention of triple-negative breast cancer cell growth and metastasis.

The Effect of Hyperthermia and Radiotherapy Sequence on Cancer Cell Death and the Immune Phenotype of Breast Cancer Cells.

Simple SummaryHyperthermia (HT) is a cancer treatment which locally heats the tumor to supraphysiological temperature, and it is an effective sensitizer for radiotherapy (RT) and chemotherapy. HT is further capable of modulating the immune system. Thus, a better understanding of its effect on the immune phenotype of tumor cells, and particularly when combined with RT, would help to optimize combined anti-cancer treatments. Since in clinics, no standards about the sequence of RT and HT exist, we analyzed whether this differently affects the cell death and immunological phenotype of human breast cancer cells. We revealed that the sequence of HT and RT does not strongly matter from the immunological point of view, however, when HT is combined with RT, it changes the immunophenotype of breast cancer cells and also upregulates immune suppressive immune checkpoint molecules. Thus, the additional application of immune checkpoint inhibitors with RT and HT should be beneficial in clinics.Hyperthermia (HT) is an accepted treatment for recurrent breast cancer which locally heats the tumor to 39–44 °C, and it is a very potent sensitizer for radiotherapy (RT) and chemotherapy. However, currently little is known about how HT with a distinct temperature, and particularly, how the sequence of HT and RT changes the immune phenotype of breast cancer cells. Therefore, human MDA-MB-231 and MCF-7 breast cancer cells were treated with HT of different temperatures (39, 41 and 44 °C), alone and in combination with RT (2 × 5 Gy) in different sequences, with either RT or HT first, followed by the other. Tumor cell death forms and the expression of immune checkpoint molecules (ICMs) were analyzed by multicolor flow cytometry. Human monocyte-derived dendritic cells (moDCs) were differentiated and co-cultured with the treated cancer cells. In both cell lines, RT was the main stressor for cell death induction, with apoptosis being the prominent cell death form in MCF-7 cells and both apoptosis and necrosis in MDA-MB-231 cells. Here, the sequence of the combined treatments, either RT or HT, did not have a significant impact on the final outcome. The expression of all of the three examined immune suppressive ICMs, namely PD-L1, PD-L2 and HVEM, was significantly increased on MCF-7 cells 120 h after the treatment of RT with HT of any temperature. Of special interest for MDA-MB-231 cells is that only combinations of RT with HT of both 41 and 44 °C induced a significantly increased expression of PD-L2 at all examined time points (24, 48, 72, and 120 h). Generally, high dynamics of ICM expression can be observed after combined RT and HT treatments. There was no significant difference between the different sequences of treatments (either HT + RT or RT + HT) in case of the upregulation of ICMs. Furthermore, the co-culture of moDCs with tumor cells of any treatment had no impact on the expression of activation markers. We conclude that the sequence of HT and RT does not strongly affect the immune phenotype of breast cancer cells. However, when HT is combined with RT, it results in an increased expression of distinct immune suppressive ICMs that should be considered by including immune checkpoint inhibitors in multimodal tumor treatments with RT and HT. Further, combined RT and HT affects the immune system in the effector phase rather than in the priming phase.

Cooperative interaction between ER\u03b1 and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

The epithelial to mesenchymal transition (EMT) has been proposed to contribute to the metastatic spread of breast cancer cells. EMT-promoting transcription factors determine a continuum of different EMT states. In contrast, estrogen receptor α (ERα) helps to maintain the epithelial phenotype of breast cancer cells and its expression is crucial for effective endocrine therapies. Determining whether and how EMT-associated transcription factors such as ZEB1 modulate ERα signaling during early stages of EMT could promote the discovery of therapeutic approaches to suppress metastasis. Here we show that, shortly after induction of EMT and while cells are still epithelial, ZEB1 modulates ERα-mediated transcription induced by estrogen or cAMP signaling in breast cancer cells. Based on these findings and our ex vivo and xenograft results, we suggest that the functional interaction between ZEB1 and ERα may alter the tissue tropism of metastatic breast cancer cells towards bone.

SEC14L3 plays a tumor-suppressive role in breast cancer through a Wnt/β-catenin-related way

Breast cancer, the most prevalent malignancy in women, is also the leading cause of cancer-related deaths in women worldwide. The activation of the Wnt pathway plays a pivotal role in the metastatic abilities of breast cancer. In this study, IL1F6, MRGPRX1, and SEC14L3 were significantly correlated to breast cancer patients'overall survival based on TCGA-BRCA dataset. Although IL1F6, MRGPRX1 and SEC14L3 high expression were associated with better survival in breast cancer patients, SEC14L3 had the biggest survival benefit for breast cancer; therefore, SEC14L3 was selected for the subsequent investigation. SEC14L3 mRNA expression and protein levels within breast cancer cell lines decreased compared with normal human breast epithelial cells. Overexpressing SEC14L3 in breast cancer cells inhibited the malignant phenotypes of cancer cells, including the capacity of cells to migrate and invade. SEC14L3 overexpression decreased the levels of mesenchymal markers, whereas SEC14L3 knockdown facilitated the malignant behaviors of breast cancer cells. SEC14L3 overexpression also inhibited Wnt/β-catenin activation. The Wnt agonist strengthened the malignant phenotypes of breast cancer cells; moreover, the anti-tumor effects of SEC14L3 overexpression were partially attenuated by the Wnt agonist. Conclusively, SEC14L3, which is underexpressed in breast cancer cells and tissues, could play a tumor-suppressive role in a Wnt/β-catenin-related way.

The lactate sensor GPR81 regulates glycolysis and tumor growth of breast cancer

Metabolic reprogramming is a malignant phenotype of cancer. Cancer cells utilize glycolysis to fuel rapid proliferation even in the presence of oxygen, and elevated glycolysis is coupled to lactate fermentation in the cancer microenvironment. Although lactate has been recognized as a metabolic waste product, it has become evident that lactate functions as not only an energy source but a signaling molecule through the lactate receptor G-protein-coupled receptor 81 (GPR81) under physiological conditions. However, the pathological role of GPR81 in cancer remains unclear. Here, we show that GPR81 regulates the malignant phenotype of breast cancer cell by reprogramming energy metabolism. We found that GPR81 is highly expressed in breast cancer cell lines but not in normal breast epithelial cells. Knockdown of GPR81 decreased breast cancer cell proliferation, and tumor growth. Mechanistically, glycolysis and lactate-dependent ATP production were impaired in GPR81-silenced breast cancer cells. RNA sequencing accompanied by Gene Ontology enrichment analysis further demonstrated a significant decrease in genes associated with cell motility and silencing of GPR81 suppressed cell migration and invasion. Notably, histological examination showed strong expression of GPR81 in clinical samples of human breast cancer. Collectively, our findings suggest that GPR81 is critical for malignancy of breast cancer and may be a potential novel therapeutic target for breast carcinoma.

Abstract P5-11-02: Humoral immune responses direct breast cancer towards a basal-like phenotype and increases stemness and invasive potential

Abstract Introduction: A large subset of triple negative breast cancers (TNBC) are characterized by a high degree of lymphocytic infiltration. Previous studies have shown that TNBC contain higher densities of B lymphocytes than other breast cancer subtypes. B lymphocytes infiltration occurs early in breast cancer and is associated with microinvasion. Although the exact role of B cell in growth of TNBC is unclear, B cells have been shown to induce chronic inflammatory responses in other types of cancer such as melanoma through the activation of NF-KB. Chronic inflammation is known to increase stem cell features and promote epithelial mesenchymal transition (EMT) in cancer. Therefore, we sought to examine interactions between B cells and TNBC and determine the effects on stemness and phenotype of breast cancer cells. Methods: CD20 expression was evaluated in tissue microarrays of ER- DCIS by immunohistochemistry. To mimic the B cell rich tumor microenvironment, co-culture studies were performed with the TNBC cell line SUM159 and EBV-transformed B cells using FalconTM Cell Culture Insert with a 0.4 um filter. Expression of stemness genes in tumor cells after co-culture was evaluated using Human pluripotent stem cell kit array (Qiagen). Expression of EMT and stemness genes and markers were determined by qPCR and western blotting. Self-renewal was assessed using mammosphere assays of co-cultured cells compared to controls. Invasion assays were performed in SUM159 and MCF-12A cell lines after co-culture using Corning® BiocoatTM cell culture inserts. Results: High densities of B cells were found in 9 of 36 DCIS (25%) and associated with microinvasion (p=.035). A 2-fold increase in invasion was observed in co-cultured cells compared to controls. RTqPCR showed increased expression of stemness genes OCT4 and Nanog (1.3- and 2.3-fold change respectively). Additionally, increased protein expression of stemness genes (Snail, SOX17, α-Fetoprotein AFP, Otx2, Nanog, Sox2, Oct4 and pdx-1) was observed in co-cultured cells compared to the control in the commercial array analysis. Likewise, we observed a 2- fold increase in ALDH1A3 gene expression which correlated with an increase in ALDH1A3 protein expression in co-cultured cells compared to controls. Additionally, co-cultured cells exhibited a two-fold increase in mammosphere formation compared to controls indicating an increase in self-renewal. A 7-fold increase in gene expression in the mesenchymal marker vimentin and a slight increase expression of snail was observed. This change correlated with a decrease in gene and protein expression of E-cadherin indicating an increase in EMT in co-cultured cells. Conclusion: Our study confirms the finding that B cell infiltration in stage 0 breast cancer is associated to microinvasion. Further, our in vitro studies indicate that interactions between B cells and TNBC may lead to increased invasion, stemness, and EMT. Thus, early B cell infiltration may be a driver of aggressive disease. Further study is needed to examine cross-talk between B cells and TNBC which may identify new targets for therapeutic intervention. Additionally, studies should examine CD20+ B cell infiltration as a prognostic marker for ER- DCIS progression. Citation Format: Jennifer Sims Mourtada, Steficah Maosa, Lynn Opdenaker, Nicole Toney. Humoral immune responses direct breast cancer towards a basal-like phenotype and increases stemness and invasive potential [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-11-02.

Abstract P1-05-09: Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method

Abstract Purpose: Immunophenotyping is a useful tool for diagnostic and therapeutic assessment of heterogenous breast cancer cells that depend on external factors and growth conditions. The objective of this study was to characterize the phenotype of patient-derived breast cancer cells (PDBCCs) grown in different culture medium and under different conditions including 2D monolayer and non-adherent 3D spheroid culture by using flow cytometry. Methods: PDBCCs and normal breast cells were harvested from fresh surgical specimens (N=11) of breast tumor and normal tissues. PDBCCs were grown in various culture media under monolayer, spheroid and organoid conditions. Flow cytometry, qRT-PCR, ALDEFLUOR assay, β-galactosidase staining and RNA-seq were performed. Results: PDBCC growth rates were different for each patient case. As compared with full media, A83-01 removal increased EpCAM+CD24+ cells (1.8-fold, p<0.029) and decreased EpCAM-CD44high/+CD24- (0.45-fold, p=0.029). EGF removal increased EpCAM+CD49f− cells (14.1-fold, p=0.029). Removal of both EGF and A83-01 induced senescent cells with high β-galactosidase activity. EpCAM-CD44high/+CD24- cells, ALDHhigh cells, and EpCAMlow/-CD49f+ cells were enriched by 3D spheroids relative to 2D monolayer. 562 genes with 2-fold change and, p-value<0.05 were differentially expressed in 3D spheroids compared to 2D monolayers. Based on public database, many genes associated with poor prognosis in 3D spheroids were identified (Table 1). Table 1. Clinically poor prognosis-associated DEGs were selected from tumor spheroids based on recurrence-free survival analysis using Kaplan-Meier Plotter on database that includes estrogen receptor positive patients. B, coefficient of regression; HR, hazard ratio; CI, confidence interval; ER, estrogen receptor, HER2, human epidermal receptor 2; RFS, recurrent free survival; FC, fold change. Conclusion: PDBCC phenotypes can be modulated by external factors and culture methods. Cancer stem-like properties of PDBCCs can be enriched by 3D spheroids. Future studies to explore the specific molecular mechanisms underlying these observations may provide new molecular targets for the development of therapies for heterogenous breast cancer Clinically poor prognosis-associated DEGs based on recurrence-free survival analysisGenes symbolLog2(FC) P<0.05Prognosis95% CI for Exp(B)HRlowerupperP-valueTop 30 of DEGsUpregulated genesMMP110.90Poor1.611.381.877.5e-10BMP68.85Poor1.21.021.040.024MYCN7.20Poor1.231.041.460.015COL22A17.12Poor1.571.172.110.0026CYP1A1(CP11)7.01Poor1.271.051.530.012PGBD56.78Poor1.221.051.420.011S100P6.70Poor1.491.281.733.3e-07CALML56.24Poor1.271.0810480.0034PPP2R2C6.19Poor1.491.131.970.005PMEPA16.07Poor1.181.011.40.043Downregulated geneFOSB-6.36Poor0.630.520.762e-06LINC00844-6.01Poor0.670.480.930.017DLK2-5.46Poor0.820.710.960.011IL33-5.31Poor0.650.550.773.1e-7SCN2B-5.09Poor0.80.670.950.0099CLDN19-4.80Poor0.660.490.890.0065FOS-4.67Poor0.670.570.798.5e-7EGR1-4.63Poor0.680.580.822.3e-0.5JAM2-4.62Poor0.80.680.950.0092MYH11-4.38Poor0.760.660.890.00045ALPL-4.35Poor0.820.710.960.015 Citation Format: Seungyeon RYU, Hoe Suk Kim, So-Hyun Yoon, Sangeun Lee, Junhyuk Song, Moonjou Baek, Han-Byoel Lee, Sangyong Jon, Wonshik Han. Analysis of immunophenotyping of patient-derived primary breast cancer cells according to the medium composition and culture method [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-05-09.

Increase in ADAR1p110 activates the canonical Wnt signaling pathway associated with aggressive phenotype in triple negative breast cancer cells

Triple-negative breast cancer (TNBC) represents a challenge in the search for new therapeutic targets. TNBCs are aggressive and generate resistance to chemotherapy. Tumors of TNBC patients with poor prognosis present a high level of adenosine deaminase acting on RNA1 (ADAR1). We explore the connection of ADAR1 with the canonical Wnt signaling pathway and the effect of modulation of its expression in TNBC. Expression data from cell line sequencing (DepMap) and TCGA samples were downloaded and analyzed. We lentivirally generated an MDA-MB-231 breast cancer cell line that overexpress (OE) ADAR1p110 or an ADAR knockdown. Abundance of different proteins related to Wnt/β-catenin pathway and activity of nuclear β-catenin were analyzed by Western blot and luciferase TOP/FOP reporter assay, respectively. Cell invasion was analyzed by matrigel assay. In mice, we study the behavior of tumors generated from ADAR1p110 (OE) cells and tumor vascularization immunostaining were analyzed. ADAR1 connects to the canonical Wnt pathway in TNBC. ADAR1p110 overexpression decreased GSK-3β, while increasing active β-catenin. It also increased the activity of nuclear β-catenin and increased its target levels. ADAR1 knockdown has the opposite effect. MDA-MB-231 ADAR1 (OE) cells showed increased capacity of invasion. Subsequently, we observed that tumors derived from ADAR1p110 (OE) cells showed increased invasion towards the epithelium, and increased levels of Survivin and CD-31 expressed in vascular endothelial cells. These results indicate that ADAR1 overexpression alters the expression of some key components of the canonical Wnt pathway, favoring invasion and neovascularization, possibly through activation of the β-catenin, which suggests an unknown role of ADAR1p110 in aggressiveness of TNBC tumors.