Nucleus Of Breast Cancer Cells Research Articles

Artemisitene induces apoptosis of breast cancer cells by targeting FDFT1 and inhibits the growth of breast cancer patient-derived organoids

Artemisitene (ATT) is a natural bioactive compound with anti-breast cancer activity. However, the direct target and clinical efficacy of ATT on breast cancer are still unclear. The current study aimed to identify the target protein and underlying mechanism of ATT in anti-breast cancer. Moreover, patient-derived organoids (PDOs) were employed to assess the clinical efficacy of ATT on breast cancer. Herein, molecular docking, molecular dynamics simulation, cellular thermal shift assay (CETSA) combined with Western blot, surface plasmon resonance (SPR) were applied to confirm the interactional target of ATT in breast cancer cells. Bioinformatics analysis, Western blot, flow cytometry, plasmid construction and lentivirus infection, chromatin immunoprecipitation (ChIP) assay, and quantitative real-time PCR (RT-qPCR) were performed to reveal the potential mechanism of ATT in treating breast cancer. PDOs were established to evaluate the clinical therapeutic efficiency of ATT on breast cancer. We found that ATT interacted with Farnesyl-diphosphate farnesyltransferase 1 (FDFT1) in breast cancer cells. Knockdown of FDFT1 induced NEDD4 expression and apoptosis in breast cancer cells. Overexpression of FDFT1 could rescue ATT-induced apoptosis, while interfering with FDFT1 expression decreased the level of RelA (NF-κB p65 subunit) in the nucleus in breast cancer cells. Knockdown of FDFT1 induced NEDD4 expression by regulating TNFR1/NF-κB pathway. Overexpression of FDFT1 could reverse the activation of ATT-induced TNFR1/NF-κB/NEDD4 pathway in breast cancer cells. Interestingly, the ChIP assay and RT-qPCR revealed that p65 could regulate NEDD4 transcription. Furthermore, ATT exhibited a broad-spectrum inhibitory effect on the growth of breast cancer PDOs with different pathological subtypes, and showed an excellent safety profile in comparison with that of conventional chemotherapy drugs. In summary, this work demonstrated that ATT targets FDFT1 to induce apoptosis of breast cancer cells through regulating TNFR1/NF-κB/NEDD4 pathway and suppresses breast cancer PDOs growth, which supported ATT as an effective and potential drug candidate for breast cancer treatment.

AKT-dependent nuclear localization of EPRS1 activates PARP1 in breast cancer cells

Glutamyl-prolyl-tRNA synthetase (EPRS1) is a bifunctional aminoacyl-tRNA-synthetase (aaRS) essential for decoding the genetic code. EPRS1 resides, with seven other aaRSs and three noncatalytic proteins, in the cytoplasmic multi-tRNA synthetase complex (MSC). Multiple MSC-resident aaRSs, including EPRS1, exhibit stimulus-dependent release from the MSC to perform noncanonical activities distinct from their primary function in protein synthesis. Here, we show EPRS1 is present in both cytoplasm and nucleus of breast cancer cells with constitutively low phosphatase and tensin homolog (PTEN) expression. EPRS1 is primarily cytosolic in PTEN-expressing cells, but chemical or genetic inhibition of PTEN, or chemical or stress-mediated activation of its target, AKT, induces EPRS1 nuclear localization. Likewise, preferential nuclear localization of EPRS1 was observed in invasive ductal carcinoma that were also P-Ser473-AKT+. EPRS1 nuclear transport requires a nuclear localization signal (NLS) within the linker region that joins the catalytic glutamyl-tRNA synthetase and prolyl-tRNA synthetase domains. Nuclear EPRS1 interacts with poly(ADP-ribose) polymerase 1 (PARP1), a DNA-damage sensor that directs poly(ADP-ribosyl)ation (PARylation) of proteins. EPRS1 is a critical regulator of PARP1 activity as shown by markedly reduced ADP-ribosylation in EPRS1 knockdown cells. Moreover, EPRS1 and PARP1 knockdown comparably alter the expression of multiple tumor-related genes, inhibit DNA-damage repair, reduce tumor cell survival, and diminish tumor sphere formation by breast cancer cells. EPRS1-mediated regulation of PARP1 activity provides a mechanistic link between PTEN loss in breast cancer cells, PARP1 activation, and cell survival and tumor growth. Targeting the noncanonical activity of EPRS1, without inhibiting canonical tRNA ligase activity, provides a therapeutic approach potentially supplementing existing PARP1 inhibitors.

ACSL3 regulates breast cancer progression via lipid metabolism reprogramming and the YES1/YAP axis.

Mitochondrial fatty acid oxidation is a metabolic pathway whose dysregulation is recognized as a critical factor in various cancers, because it sustains cancer cell survival, proliferation, and metastasis. The acyl-CoA synthetase long-chain (ACSL) family is known to activate long-chain fatty acids, yet the specific role of ACSL3 in breast cancer has not been determined. We assessed the prognostic value of ACSL3 in breast cancer by using data from tumor samples. Gain-of-function and loss-of-function assays were also conducted to determine the roles and downstream regulatory mechanisms of ACSL3 in vitro and in vivo. ACSL3 expression was notably downregulated in breast cancer tissues compared with normal tissues, and this phenotype correlated with improved survival outcomes. Functional experiments revealed that ACSL3 knockdown in breast cancer cells promoted cell proliferation, migration, and epithelial-mesenchymal transition. Mechanistically, ACSL3 was found to inhibit β-oxidation and the formation of associated byproducts, thereby suppressing malignant behavior in breast cancer. Importantly, ACSL3 was found to interact with YES proto-oncogene 1, a member of the Src family of tyrosine kinases, and to suppress its activation through phosphorylation at Tyr419. The decrease in activated YES1 consequently inhibited YAP1 nuclear colocalization and transcriptional complex formation, and the expression of its downstream genes in breast cancer cell nuclei. ACSL3 suppresses breast cancer progression by impeding lipid metabolism reprogramming, and inhibiting malignant behaviors through phospho-YES1 mediated inhibition of YAP1 and its downstream pathways. These findings suggest that ACSL3 may serve as a potential biomarker and target for comprehensive therapeutic strategies for breast cancer.

Mechanical comparison of isolated and living breast cancer cell nuclei

Mechanical comparison of isolated and living breast cancer cell nuclei

Progesterone receptor-Grb2 interaction is associated with better outcomes in breast cancer

In addition to mediating nuclear transcription, PR mediates extranuclear functions mainly through the PR polyproline domain (PPD) interaction with the SH3 domain of cytoplasmic signaling molecules. PR-PPD-SH3 interaction inhibits EGF-mediated signaling and decreases lung cancer cell proliferation. Grb2 is an essential adaptor molecule with an SH2 domain flanked by two SH3 domains. In this study, we examined whether PR, through interaction between PR-PPD and Grb2-SH3, can interact with Grb2 in cells and breast cancer tissues. Our previous study shows that interaction between PR-PPD and Grb2 could interfere with cytoplasmic signaling and lead to inhibition of EGF-mediated signaling. GST-pulldown analysis shows that PR-PPD specifically interacts with the SH3 domains of Grb2. Immunofluorescence staining shows colocalization of PR and Grb2 in both the nucleus and cytoplasm in BT-474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we show that PR and Grb2 interact in breast cancer cells through the Grb2-SH3 domain. Proximity Ligation Assay (PLA) analysis of 43 breast cancer specimens shows that PR-Grb2 interaction is associated with low histological stage and negatively correlates with lymph node invasion and metastasis in breast cancer. These results, together with our previous findings, suggest that PR-PPD interaction with Grb2 plays an essential role in PR-mediated growth factor signaling inhibition and could contribute significantly to better prognosis in PR- and Grb2-positive breast cancer. Our finding provides a basis for additional studies to explore a novel therapeutic strategy for cancer treatment.

Cathepsin V regulates cell cycle progression and histone stability in the nucleus of breast cancer cells.

Introduction: We previously identified that Cathepsin V (CTSV) expression is associated with poor prognosis in ER+ breast cancer, particularly within the Luminal A subtype. Examination of the molecular role of the protease within Luminal A tumours, revealed that CTSV promotes tumour cell invasion and proliferation, in addition to degradation of the luminal transcription factor, GATA3, via the proteasome. Methods: Cell line models expressing CTSV shRNA or transfected to overexpress CTSV were used to examine the impact of CTSV on cell proliferation by MTT assay and flow cytometry. Western blotting analysis was used to identify the impact of CTSV on histone and chaperone protein expression. Cell fractionation and confocal microscopy was used to illustrate the presence of CTSV in the nuclear compartment. Results: In this work we have identified that CTSV has an impact on breast cancer cell proliferation, with CTSV depleted cells exhibiting delayed progression through the G2/M phase of the cell cycle. Further investigation has revealed that CTSV can control nuclear expression levels of histones H3 and H4 via regulating protein expression of their chaperone sNASP. We have discovered that CTSV is localised to the nuclear compartment in breast tumour cells, mediated by a bipartite nuclear localisation signal (NLS) within the CTSV sequence and that nuclear CTSV is required for cell cycle progression and histone stability in breast tumour cells. Discussion: Collectively these findings support the hypothesis that targeting CTSV may have utility as a novel therapeutic target in ER+ breast cancer by impairing cell cycle progression via manipulating histone stabilisation.

Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody

BackgroundPeptidyl arginine deiminase 4 (PADI4) is a post-translational modification enzymecan that converts arginine in protein into citrulline in the presence of calcium ions, which is called citrullination. PADI4 has been reported to be expressed in the cytoplasm and nucleus in a variety of malignant tumors. Based on the GeneCards database and our previous research, it is speculated that PADI4 may also be expressed on the cell membrane. This study aimed to confirm the membrane expression of PADI4 and the effect of anti-PADI4 antibodies on cell membrane PADI4. This may be another mechanism of action of anti-PADI4 monoclonal antibodies in the treatment of breast cancer.MethodsThe subcellular localizations of PADI4 in MDA-MB-231 and MCF-7 breast cancer cells were determined by immunofluorescence, immunoelectron microscopy, and Western blot analysis. The tumor cells were treated with PADI4 antibody, and cell proliferation, migration, colony formation, apoptosis, glycolysis, and epithelial-mesenchymal transition (EMT) were measured as well as the expression of some essential tumor genes.ResultsPADI4 was not only localized in the nucleus and cytoplasm of breast cancer cells but was also detected on the cell membrane. Following PADI4 antibody treatment, cell proliferation, migration, colony formation, EMT, and ATP production through glycolysis were decreased, and the mRNA expression of MYC proto-oncogene (MYC), FAT atypical cadherin 1 (FAT1), nuclear factor kappa B subunit 1 (NFκB), and tumor necrosis factor (TNF-α) in breast cancer cells was downregulated, while the mRNA expression of tumor protein p63 (TP63) was upregulated.ConclusionsPADI4 is expressed on the cell membrane in breast cancer cells. Anti-PADI4 antibodies can affect the biological functions of cell membrane PADI4, including proliferation, migration, apoptosis, and glycolysis, thereby inhibiting tumor progression.Graphical abstract

Phosphorylation of EZH2 differs HER2-positive breast cancer invasiveness in a site-specific manner

HER2-positive breast cancer (BC) invasiveness and drug-resistance issue is the critical treatment obstacle recently. We investigated the total and phosphorylated status EZH2 expression in database and BC tissue microarray. We demonstrated for the first time that EZH2 is distributed both in cytoplasm and nucleus of breast cancer cells in a phosphorylation site-specific manner. High expressed-EZH2 cases more frequently had an advanced clinical stage (lymph node metastasis) and aggressive features than EZH2-low cases, potentially indicating the high risk of HER2-positive BC (p < 0.05). Notably, highly expressed phosphorylated EZH2 is differently located in cytoplasm or nucleus in a site-specific manner in breast cancer cells. Nucleus-located pEZH2-S21 is expressed in invasive and lymph node metastatic HER2-positive BC cases (p = 0.144, p = 0.001). Cytoplasmic pEZH2-T487 is correlated with HER2 positive status (p = 0.014).In conclusion, high expression of nucleus-located EZH2 might be a predictor of invasive BC. Activation of phosphorylated EZH2-S21 site in nucleus would be a potential predictor of HER2-positve BC and poor efficacy of HER2-target therapy. These results point to a PRC2-independent non-epigenetic mechanism and therapeutic strategy of EZH2 in HER2-positive BC.

OR22-05 Progesterone Receptor-Grb2 Interaction Is Associated With Better Outcomes In Breast Cancer

Abstract Disclosure: N. Wittayavimol: None. E. Iwabuchi: None. P. Pateetin: None. Y. Miki: None. Y. Onodera: None. H. Sasano: None. V. Boonyaratanakornkit: None. The presence of progesterone receptor (PR) is usually associated with better clinical outcomes for several endocrine-related cancers. However, the molecular mechanism of how PR mediates growth inhibition remains unclear. In addition to its role as a nuclear transcription, PR also possesses an extranuclear function, mediated mainly through PR polyproline domain (PPD) interaction with the SH3 domains of cytoplasmic signaling molecules. Our previous study suggested a possible role of PR-PPD in mediating SH3-domain interaction. We showed that PR-PPD and SH3 domain interaction inhibited EGF-mediated signaling and decreased lung cancer cell proliferation. An essential adapter protein that helps transduce growth factor signaling is Grb2 which has an SH2 domain flanked by two SH3 domains. In this study, we hypothesized that PR interferes with cytoplasmic signaling through interaction between PR-PPD and Grb2-SH3. We used several protein-protein interaction assays in vitro and in cells to demonstrate PR-Grb2 interaction. GST-pulldown analysis showed that PR-PPD interacts specifically with SH3 domains of Grb2. No interaction was detected when key prolines in the PR-PPD to alanines. Immunofluorescence staining showed colocalization of PR and Grb2 in both the nucleus and cytoplasm of BT474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we showed that PR and Grb2 interacted in breast cancer cells, specifically through the Grb2-SH3 domain. No BiFC signal was detected when Grb2-SH3 domains were deleted. Furthermore, we used Proximity Ligation Assay (PLA) analysis to examine protein-protein interaction of a distance less than 40nm in cells. We found PR-Grb2 interaction in PR-positive breast cancer tissue sections. No PLA signal was detected in PR-negative breast cancer cells. Importantly, we examined 43 PR-positive breast cancer specimens and showed that PR-Grb2 interaction was associated with low histological stage and negatively correlated with lymph node invasion and metastasis of breast cancer. Our results provide a molecular mechanism for how PR can inhibit growth factor signaling by interfering with Grb2 signaling and a basis for developing novel therapeutic strategies for future cancer treatment. Presentation: Saturday, June 17, 2023

Influence of chromatin remodeling molecule ARID1A on metastatic heterogeneity in triple negative breast cancer: Binding of YAP.

1085 Background: Heterogeneity is the prominent character of TNBC and one predominant cause of treatment failure. As a key nuclear SWI/SNF protein complex subunit, ARID1A plays a critical role in tumor identity determining by manipulating gene expression. The present study aimed to investigate whether and how ARID1A determined the metastatic heterogeneity of triple-negative breast cancer. Methods: This study retrospectively collected and analysed clinical and pathological data from 258 patients diagnosed with triple-negative breast cancer at Fudan University Cancer Hospital from August 2015 to June 2016 to define the prognostic ability of ARID1A.CRISPR/Cas9 or plasmids-mediated ARID1A were used to determine the ARID1A function in regulating the metastatic character in TNBC. Cell morphology, tumor invasion, and metastasis were analyzed in terms of ARID1A status both in vitro and in vivo experiments. The molecular mechanism of ARID1A determining metastasis potential of TNBC was explored by RNA-Seq and nuclear positioning. Results: ARID1A low expression was found to have independent prognostic value for poor OS (43.9 vs 53.6 months, P=0.0001) and RFS (31.0 vs 45.5 months, P=0.0029) in TNBC patients. Both nuclear and cytoplasmic protein analysis and Immunofluorescent localization assay confirm that ARID1A recruits the Hippo pathway effector YAP into nucleus in human triple negative breast cancer cells. Next we designed the YAP truncator plasmid and confirmed by Co-Immunoprecipitation that ARID1A could competitively bind to the WW domain of YAP to form the ARID1A/YAP complex. Furthermore, down-regulation of ARID1A promoted migration and invasion both in human triple negative breast cancer cells and xenograft model through Hippo/YAP signaling axis. Conclusions: Our study provides new insights into the previously unrecognized role of ARID1A in TNBC metastasis, which might provide new therapeutic options for patients with TNBC.

Chromatin Remodelling Molecule ARID1A Determines Metastatic Heterogeneity in Triple-Negative Breast Cancer by Competitively Binding to YAP.

Heterogeneity represents a pivotal factor in the therapeutic failure of triple-negative breast cancer (TNBC). In this study, we retrospectively collected and analysed clinical and pathological data from 258 patients diagnosed with TNBC at the Fudan University Cancer Hospital. Our findings show that low ARID1A expression is an independent prognostic indicator for poor overall survival (OS) and recurrence-free survival (RFS) in TNBC patients. Mechanistically, both nuclear and cytoplasmic protein analyses and immunofluorescent localisation assays confirm that ARID1A recruits the Hippo pathway effector YAP into the nucleus in human triple-negative breast cancer cells. Subsequently, we designed a YAP truncator plasmid and confirmed through co-immunoprecipitation that ARID1A can competitively bind to the WW domain of YAP, forming an ARID1A/YAP complex. Moreover, the downregulation of ARID1A promoted migration and invasion in both human triple-negative breast cancer cells and xenograft models through the Hippo/YAP signalling axis. Collectively, these findings demonstrate that ARID1A orchestrates the molecular network of YAP/EMT pathways to affect the heterogeneity in TNBC.

Binding of combined irinotecan and epicatechin to a pH-responsive DNA tetrahedron for controlled release and enhanced cytotoxicity

pH-Responsive aptamer-linked DNA nanostructures can actively target cancer cells to release drugs in the cells’ acidic environment. Tea polyphenols can be used as cancer-preventive agents to enhance the efficacy of chemotherapeutic drugs. In this study, we constructed a pH-responsive DNA tetrahedron with MUC1 aptamer (MUC1-TD) to co-load irinotecan hydrochloride (CPT-11) and (-)-epicatechin (EC). The thermodynamic parameters for the interaction of CPT-11 and/or EC with MUC1-TD were investigated using fluorescence spectroscopy and calorimetry. The binding of CPT-11 to MUC1-TD was found to be stronger than that of EC. Differential scanning calorimetry and fluorescence quenching experiments demonstrated the intercalative binding between CPT-11/EC and MUC1-TD. The in vitro release rate of CPT-11 was faster at pH 5.0 than at pH 7.4, confirming the pH-responsive of MUC1-TD. In vitro cytotoxicity results indicated that CPT-11 loaded by MUC1-TD was more cytotoxic than free CPT-11. Further, we found that the cytotoxicity of CPT-11 was further enhanced in the presence of EC due to a synergy between them. Additionally, cell uptake experiments showed that CPT-11 and EC loaded by MUC1-TD were mainly distributed in the nucleus of human breast cancer cells (MCF-7). These findings indicate that pH-responsive DNA nanostructures are a promising delivery system for co-loading chemotherapeutic drugs and natural polyphenols.

Intracellular and extracellular moesins differentially regulate Src activity and β-catenin translocation to the nucleus in breast cancer cells

It is increasingly recognized that a single protein can have multiple, sometimes paradoxical, roles in cell functions as well as pathological conditions depending on its cellular locations. Here we report that moesins (MSNs) in the intracellular and extracellular domains present opposing roles in pro-tumorigenic signaling in breast cancer cells. Using live cell imaging with fluorescence resonance energy transfer (FRET)- and green fluorescent protein (GFP)-based biosensors, we investigated the molecular mechanism underlying the cellular location-dependent effect of MSN on Src and β-catenin signaling in MDA-MB-231 breast cancer cells. Inhibition of intracellular MSN decreased the activities of Src and FAK, whereas overexpression of intracellular MSN increased them. By contrast, extracellular MSN decreased the activities of Src, FAK, and RhoA, as well as β-catenin translocation to the nucleus. Consistently, Western blotting and MTT-based analysis showed that overexpression of intracellular MSN elevated the expression of oncogenic genes, such as p-Src, β-catenin, Lrp5, MMP9, Runx2, and Snail, as well as cell viability, whereas extracellular MSN suppressed them. Conditioned medium derived from MSN-overexpressing mesenchymal stem cells or osteocytes showed the anti-tumor effects by inhibiting the Src activity and β-catenin translocation to the nucleus as well as the activities of FAK and RhoA and MTT-based cell viability. Conditioned medium derived from MSN-inhibited cells increased the Src activity, but it did not affect the activities of FAK and RhoA. Silencing CD44 and/or FN1 in MDA-MB-231 cells blocked the suppression of Src activity and β-catenin accumulation in the nucleus by extracellular MSN. Collectively, the results suggest that cellular location-specific MSN is a strong regulator of Src and β-catenin signaling in breast cancer cells, and that extracellular MSN exerts tumor-suppressive effects via its interaction with CD44 and FN1.

HIV Tat-Conjugated Histone H3 Peptides Induce Tumor Cell Death Via Cellular Stress Responses.

Histone H3 is a nucleosome scaffold protein that is involved in a variety of intracellular processes. Aberrant modification of H3 is important in carcinogenesis. In contrast, free histones in cells can act as stimuli to trigger cellular immune responses and cell death. In this study, we linked cell-penetrating peptide HIV Tat to a histone H3 fragment to achieve intracellular delivery in tumor cells. We found that Tat-conjugated histone polypeptides localized to nuclei of lung and breast cancer cells and caused cell death. A trans-configured Tat sequence displayed dramatically improved peptide half-life and cytotoxicity. Mechanistic studies demonstrated that treatment with the peptides significantly elevated mitogen-activated protein kinase (MAPK) signaling, reactive oxygen species (ROS) production, as well as levels of stress-inducible transcription factor ATF3 (activating transcription factor 3) and AP-1 (activating protein-1). Cytotoxicity of the peptide was significantly reduced by inhibition of AP-1 activity and ROS production. These results suggest the potential of Tat-conjugated H3 peptides as antitumor agents to induce cell death via increased cellular stress response by activating p38-MAPK signaling and intracellular ROS production.

A simple, robust and scalable route to prepare sub-50 nm soft PDMS nanoparticles for intracellular delivery of anticancer drugs

Soft nanoparticles (NPs) have recently emerged as a promising material for intracellular drug delivery. In this regard, NPs derived from polydimethylsiloxane (PDMS), an FDA approved polymer can be a suitable alternative to conventional soft NPs due to their intrinsic organelle targeting ability. However, the available synthesis methods of PDMS NPs are complicated or require inorganic fillers, forming composite NPs and compromising their native softness. Herein, for the first time, we present a simple, robust and scalable strategy for preparation of virgin sub-50 nm PDMS NPs at room temperature. The NPs are soft in nature, hydrophobic and about 30 nm in size. They are stable in physiological medium for two months and biocompatible. The NPs have been successful in delivering anticancer drug doxorubicin to mitochondria and nucleus of cervical and breast cancer cells with more than four-fold decrease in IC50 value of doxorubicin as compared to its free form. Furthermore, evaluation of cytotoxicity in reactive oxygen species detection, DNA fragmentation, apoptosis-associated gene expression and tumor spheroid growth inhibition demonstrate the PDMS NPs to be an excellent candidate for delivery of anticancer drugs in mitochondria and nucleus of cancer cells.

Detection and Spatiotemporal Analysis of In-vitro 3D Migratory Triple-Negative Breast Cancer Cells.

The invasion of cancer cells into the surrounding tissues is one of the hallmarks of cancer. However, a precise quantitative understanding of the spatiotemporal patterns of cancer cell migration and invasion still remains elusive. A promising approach to investigate these patterns are 3D cell cultures, which provide more realistic models of cancer growth compared to conventional 2D monolayers. Quantifying the spatial distribution of cells in these 3D cultures yields great promise for understanding the spatiotemporal progression of cancer. In the present study, we present an image processing and segmentation pipeline for the detection of 3D GFP-fluorescent triple-negative breast cancer cell nuclei, and we perform quantitative analysis of the formed spatial patterns and their temporal evolution. The performance of the proposed pipeline was evaluated using experimental 3D cell culture data, and was found to be comparable to manual segmentation, outperforming four alternative automated methods. The spatiotemporal statistical analysis of the detected distributions of nuclei revealed transient, non-random spatial distributions that consisted of clustered patterns across a wide range of neighbourhood distances, as well as dispersion for larger distances. Overall, the implementation of the proposed framework revealed the spatial organization of cellular nuclei with improved accuracy, providing insights into the 3 dimensional inter-cellular organization and its progression through time.

Abstract 2369: The NRF2 and dimeric STAT3 complex coordinately regulates IL-23 expression: Implications for breast cancer progression

Abstract Basal-like breast cancer (BLBC) is an aggressive malignancy with a poor prognosis. Persistent activation of NRF2 and STAT3 is considered to stimulate the aggressive behavior of BLBC. An online database, NRF2-ome predicted the possibility of interplay between NRF2 and STAT3 based on domain motif interactions. However, cooperation between NRF2 and STAT3 and its implications for breast cancer progression remain elusive. Immunoprecipitation and in situ proximity analyses revealed that NRF2 and STAT3 directly interact with each other, which predominantly occurred in the nucleus of human breast cancer cells. After confirming the physical interaction between STAT3 and NRF2, we examined whether this could influence the expression or stability of each transcription factor. While silencing of NRF2 failed to alter the STAT3 expression, STAT3 knockdown facilitated degradation of NRF2. Of note, silencing of STAT3 did not affect the mRNA expression of NRF2, suggesting that STAT3-induced accumulation of NRF2 is attributable to stabilization of the preexisting protein. As the NRF2-STAT3 complex was found to accumulate predominantly in the nucleus, we speculate that NRF2 could bind to a functionally active phosphorylated form of STAT3 (P-STAT3Y705). The substitution of Y705 of STAT3 with the non-phosphorylatable phenylalanine (STAT3Y705F) abolished interaction with NRF2. Notably, the dimeric form, but not a monomeric form, of STAT3 interacts directly with the Neh1 and Neh3 domains of NRF2. Next, we investigated the functional significance of STAT3- NRF2 interaction in BLBC growth in vivo. STAT3 knockdown significantly reduced expression of NRF2 in the xenograft tumor tissues derived from MDA-MB-231 human breast cancer cells. Tumors derived from cells transfected with the non-specific control siRNA showed a pronounced interaction between STAT3 and NRF2, whereas such interaction was abolished in the STAT3-silenced xenograft tumor. Analysis of the TCGA multi-omics data showed that high levels of STAT3 and NRF2 mRNA transcripts were correlated with elevated expression of P-STAT3Y705 and NRF2 target proteins in breast cancer patients. Next generation RNA sequencing analysis identified the gene encoding interleukin-23A (IL-23A) upregulated by concurrent binding of STAT3 and NRF2 to its promoter. IL-23A is markedly overexpressed in BLBC, compared with other subtypes of breast cancer. Combined silencing of NRF2 and STAT3 inhibited BLBC growth to a greater extent than that achieved with single siRNA transfection. IL-23A depletion by siRNA also showed the similar phenotypic changes to those caused by double knockdown of both transcription factors. In conclusion, these findings suggest that the STAT3-NRF2 interaction accelerates BLBC growth and progression by augmenting IL-23A expression, which underscores the importance of subtype-specific molecular pathways in human breast cancer. Citation Format: Su-Jung Kim, Soma Saeidi, Nam-Chul Cho, Seung Hyeon Kim, Han-Byoel Lee, Wonshik Han, Hye-Kyung Na, Young-Joon Surh. The NRF2 and dimeric STAT3 complex coordinately regulates IL-23 expression: Implications for breast cancer progression [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 2369.

Altered Retrograde Signaling Patterns in Breast Cancer Cells Cybrids with H and J Mitochondrial DNA Haplogroups.

The aim of this study was to determine the role of retrograde signaling (mitochondria to nucleus) in MCF7 breast cancer cells. Therefore, in the present study, MCF7-H and MCF7-J cybrids were produced using the mitochondria from the same H and J individuals that were already used in our non-diseased retinal pigment epithelium (ARPE19) cybrids. MCF7 cybrids were treated with cisplatin and analyzed for cell viability, mitochondrial membrane potential, ROS, and expression levels of genes associated with the cGAS-STING and cancer-related pathways. Results showed that unlike the ARPE19-H and ARPE19-J cybrids, the untreated MCF7-H and MCF7-J cybrids had similar levels of ATP, lactate, and OCR: ECAR ratios. After cisplatin treatment, MCF7-H and MCF7-J cybrids showed similar (a) decreases in cell viability and ROS levels; (b) upregulation of ABCC1, BRCA1 and CDKN1A/P21; and (c) downregulation of EGFR. Cisplatin-treated ARPE19-H and ARPE19-J cybrids showed increased expression of six cGAS-STING pathway genes, while two were increased for MCF7-J cybrids. In summary, the ARPE19-H and ARPE19-J cybrids behave differentially from each other with or without cisplatin. In contrast, the MCF7-H and MCF7-J cybrids had identical metabolic/bioenergetic profiles and cisplatin responses. Our findings suggest that cancer cell nuclei might have a diminished ability to respond to the modulating signaling of the mtDNA that occurs via the cGAS-STING pathway.

Co-expression of nuclear heterogeneous nuclear ribonucleic protein K and estrogen receptor α in endometrial cancer

Heterogeneous nuclear ribonucleic protein K (hnRNPK) regulates the expression of various genes, but has contradictory roles as a tumor promoter and a tumor suppressor. We recently reported that the expression of hnRNPK is negatively associated with malignant behavior of breast cancer where it was induced by estrogen, and bound to estrogen receptor α (ERα) in the nucleus of breast cancer cells. However, the significance of hnRNPK in endometrial cancer, also an estrogen-dependent cancer, remains unclear. In this study, we first examined the localization of hnRNPK and ERα in normal endometrium and endometrial cancer. hnRNPK and ERα immunoreactivity was detected in the nuclei of endometrial glandular and carcinoma cells. In normal endometria, hnRNPK labeling index/immuno-intensity was significantly higher in the proliferative phase than in the secretory phase. In endometrial cancer tissues, hnRNPK labeling index/immuno-intensity was significantly higher in the adjacent non-malignant glandular cells compared to that in carcinoma cells. Immunohistochemistry results for ERα were identical to that of hnRNPK both in normal endometrium and endometrial cancer. In normal and cancerous tissues, the median value of the hnRNPK labeling index was significantly higher in the ERα-high group. Intratumoral estrogen, but not androgen, measured using liquid chromatography-tandem mass spectrometry, was significantly positively correlated with the hnRNPK labeling index in endometrial cancer tissues. Database analysis revealed that the hnRNPK high expression group had a significantly better prognosis for both overall and disease-free survival. These results suggest that hnRNPK interacts with ERα to regulate endometrial changes during the menstrual cycle and suppress the malignant behavior of endometrial cancer.

Endocytic protein intersectin1-S shuttles into nucleus to suppress the DNA replication in breast cancer

Breast cancer is the most common type of cancer worldwide. However, the well-known molecular biomarkers are not enough to meet the needs of precision medicine. In search for novel targets in this regard, we reported ITSN1 (intersectin1) as one of the candidates through mRNA microarray analysis. In the present study, we reported that endocytic protein ITSN1-S exists not only in the cytoplasm but also in nuclei of breast cancer cells. ITSN1-S′ functional nuclear localization signal is within its residues 306–312. Its nuclear export signal (NES) resides within its SH3 domains. We also found, the interaction between the CC domain of nuclear ITSN1-S and the NT domain of nuclear DNA helicase II (NDH II) directly suppressed the DNA replication and nascent DNA synthesis by inhibiting the R-loops resolution in breast cancer cells. Furthermore, the interaction between the EH domains of cytoplasmic ITSN1-S and PI3KC2α inhibit cell migration and invasion by inactivating the PI3KC2α-AKT pathway. Our results were confirmed in both ITSN1 gene knockout cells and in vivo assays. Finally, our clinical data showed a potential application of the combined consideration of the cytoplasmic and nuclear ITSN1-S as an independent prognosis factor. In conclusion, our study revealed ITSN1-S′ novel positioning in the nuclei of breast cancer cells, its function in suppressing DNA replication, and its potential application in improved breast cancer prognosis.