ROR2 promotes invasion and chemoresistance of triple-negative breast cancer cells by activating PI3K/AKT/mTOR signaling.

Objective: This study aimed to investigate the role of receptor tyrosine kinase-like orphan receptor 2 (ROR2) in triple-negative breast cancer (TNBC). Methods: ROR2 expression in primary TNBC and metastatic TNBC tissues was analyzed by immunohistochemical staining and PCR. ROR2 expression in TNBC cell lines was detected by PCR and Western blot analysis. The migration, invasion and chemosensitivity of TNBC cells with overexpression or knockdown of ROR2 were examined. Results: ROR2 expression was high in metastatic TNBC tissues. ROR2 knockdown suppressed the migration, invasion and chemoresistance of TNBC cells. ROR2 overexpression in MDA-MB-435 cells promoted the migration, invasion, and chemoresistance. Moreover, ROR2 knockdown in HC1599 and MDA-MB-435 adriamycin-resistant cells enhanced chemosensitivity to adriamycin. ROR2 could activate PI3K/AKT/mTOR signaling in TNBC cells. Conclusion: ROR2 is upregulated and promotes metastatic phenotypes of TNBC by activating PI3K/AKT/mTOR signaling.

SLC7A2-Mediated Lysine Catabolism Inhibits Immunosuppression in Triple Negative Breast Cancer.

Breast cancer is one of the most common malignant tumors worldwide. SLC7A2 is abnormally expressed in multiple cancers. However, its potential in triple negative breast cancer (TNBC) is still unclear. The purpose of this study was to investigate the roles of SLC7A2 and its underlying molecular mechanisms in TNBC. mRNA expression was detected by RT-qPCR. Protein expression was detected by western blot. Co-localization of ACOX1 and TCF1 was determined using FISH assay. Histone crotonylation was performed using in vitro histone crotonylation assay. Functional analysis was performed using CCK-8 and flow cytometry assays. Xenograft assay was conducted to further verify the role of SLC7A2 in TNBC. CD8A expression was detected using immunohistochemistry. We found that SLC7A2 is downregulated in TNBC tumors. Low levels are associated with advanced stages and lymph node metastasis. SLC7A2 expression is positively correlated with CD8A. SLC7A2-mediated lysine catabolism drives the activation of CD8+ T cells. Moreover, SLC7A2 promotes histone crotonylation via upregulating ACOX1. It also promotes interaction between ACOX1 and TCF1, thus promoting antitumor T cell immunity. Additionally, overexpression of SLC7A2 activates CD8+ T cells and enhances the chemosensitivity of anti-PD-1 therapies in vivo. In conclusion, SLC7A2 may function as an antitumor gene in TNBC by activating antitumor immunity, suggesting SLC7A2/ACOX1/TCF1 signaling as a promising therapeutic strategy.

Triple-Negative Breast Cancer Subclassified by Immunohistochemistry: Correlation with Clinical and Pathological Outcomes in Patients Receiving Neoadjuvant Chemotherapy.

The intrinsic subtype of triple-negative breast cancer (TNBC) is based on genomic evaluation. In this study, we report the survival and pathological complete response (pCR) rates of TNBC patients subtyped by IHC and treated with neoadjuvant chemotherapy (NACT). A retrospective cohort of 187 TNBC patients who received NACT between 2008 and 2017 was used, and IHC subtyping was performed on biopsy specimens before chemotherapy. The subtyping revealed predominantly basal-like tumors (IHC-BL, 61%), followed by basal-like immune-suppressed tumors (IHC-BLIS, 31%), mesenchymal tumors (12.5%), luminal androgen receptor tumors (IHC-LAR, 12%), and basal-like immune-activated tumors (IHC-BLIA, 10.9%). The pCR rate varied among subtypes, with IHC-BLIA showing the highest (30.0%) and IHC-LAR showing the lowest (4.5%). IHC-BLIS led in recurrence sites. Overall and disease-free survival analyses did not show significant differences among subtypes, although IHC-BLIA demonstrated a trend toward better survival, and IHC-mesenchymal, worse. Patients who achieved pCR exhibited significantly better disease-free survival and overall survival than non-responders. This study underscores the potential of IHC-based subtyping in TNBC management, highlighting distinct response patterns to neoadjuvant chemotherapy and potential implications for treatment strategies. Further research is warranted to validate these findings and explore tailored therapeutic approaches for specific TNBC subtypes.

Breast Cancer Plasticity after Chemotherapy Highlights the Need for Re-Evaluation of Subtyping in Residual Cancer and Metastatic Tissues.

This research paper presents a novel approach to identifying biomarkers that can be used to prognosticate patients with triple-negative breast cancer (TNBC) eligible for neoadjuvant therapy. The study utilized survival and RNA sequencing data from a cohort of TNBC patients and identified 276 genes whose expression was related to survival in such patients. The gene expression data were then used to classify patients into two major groups based on the presence or absence of Wingless/Integrated-pathway (Wnt-pathway) and mesenchymal (Mes) markers (Wnt/Mes). Patients with a low expression of Wnt/Mes-related genes had a favorable outcome, with no deaths observed during follow-up, while patients with a high expression of Wnt/Mes genes had a higher mortality rate of 50% within 19 months. The identified gene list could be validated and potentially used to shape treatment options for TNBC patients eligible for neoadjuvant therapy providing valuable insights into the development of more effective treatments for TNBC. Our data also showed significant variation in gene expression profiles before and after chemotherapy, with most tumors switching to a more mesenchymal/stem cell-like profile. To verify this observation, we performed an in silico analysis to classify breast cancer tumors in Prediction Analysis of Microarray 50 (PAM50) molecular classes before treatment and after treatment using gene expression data. Our findings demonstrate that following drug intervention and metastasis, certain tumors undergo a transition to alternative subtypes, resulting in diminished therapeutic efficacy. This underscores the necessity for reevaluation of patients who have experienced relapse or metastasis post-chemotherapy, with a focus on molecular subtyping. Tailoring treatment strategies based on these refined subtypes is imperative to optimize therapeutic outcomes for affected individuals.

Shikonin blocks CAF-induced TNBC metastasis by suppressing mitochondrial biogenesis through GSK-3ß/NEDD4-1 mediated phosphorylation-dependent degradation of PGC-1α.

BACKGROUND: Triple-negative breast cancer (TNBC) is characterized by its high metastatic potential, which results in poor patient survival. Cancer-associated fibroblasts (CAFs) are crucial in facilitating TNBC metastasis via induction of mitochondrial biogenesis. However, how to inhibit CAF-conferred mitochondrial biogenesis is still needed to explore. METHODS: We investigated metastasis using wound healing and cell invasion assays, 3D-culture, anoikis detection, and NOD/SCID mice. Mitochondrial biogenesis was detected by MitoTracker green FM staining, quantification of mitochondrial DNA levels, and blue-native polyacrylamide gel electrophoresis. The expression, transcription, and phosphorylation of peroxisome-proliferator activated receptor coactivator 1α (PGC-1α) were detected by western blotting, chromatin immunoprecipitation, dual-luciferase reporter assay, quantitative polymerase chain reaction, immunoprecipitation, and liquid chromatography-tandem mass spectrometry. The prognostic role of PGC-1α in TNBC was evaluated using the Kaplan-Meier plotter database and clinical breast cancer tissue samples. RESULTS: We demonstrated that PGC-1α indicated lymph node metastasis, tumor thrombus formation, and poor survival in TNBC patients, and it was induced by CAFs, which functioned as an inducer of mitochondrial biogenesis and metastasis in TNBC. Shikonin impeded the CAF-induced PGC-1α expression, nuclear localization, and interaction with estrogen-related receptor alpha (ERRα), thereby inhibiting PGC-1α/ERRα-targeted mitochondrial genes. Mechanistically, the downregulation of PGC-1α was mediated by synthase kinase 3ß-induced phosphorylation of PGC-1α at Thr295, which associated with neural precursor cell expressed developmentally downregulated 4e1 recognition and subsequent degradation by ubiquitin proteolysis. Mutation of PGC-1α at Thr295 negated the suppressive effects of shikonin on CAF-stimulated TNBC mitochondrial biogenesis and metastasis in vitro and in vivo. CONCLUSIONS: Our findings indicate that PGC-1α is a viable target for blocking TNBC metastasis by disrupting mitochondrial biogenesis, and that shikonin merits potential for treatment of TNBC metastasis as an inhibitor of mitochondrial biogenesis through targeting PGC-1α.

Eribulin induces micronuclei and enhances the nuclear localization of cGAS in triple-negative breast cancer cells.

Eribulin (ERI), clinically utilized for locally advanced or metastatic breast tumors, has shown potential links to the immune system. Notably, the cGAS-STING pathway, a key component of innate immunity, has gained prominence. Yet, limited reports explore ERI's effects on the cGAS-STING pathway. Additionally, the nuclear presence of cGAS remains poorly understood. This study uniquely delves into ERI's impact on both the cytosolic cGAS-STING pathway and nuclear cGAS. ERI enhances nuclear localization of cGAS, resulting in hyper-activation of the cGAS-STING pathway in triple-negative breast cancer cells. Reduction of cGAS heightened both cell proliferation and ERI sensitivity. In clinical data using ERI in a neo-adjuvant setting, patients with low cGAS cases exhibited reduced likelihood of achieving pathological complete response after ERI treatment. These findings illuminate the potential of cGAS and IFNß as predictive biomarkers for ERI sensitivity, providing valuable insights for personalized breast cancer treatment strategies.

Prognostic potential of CUL3 ligase with differential roles in luminal A and basal type breast cancer tumors.

Breast cancer is a prevalent and significant cause of mortality in women, and manifests as six molecular subtypes. Its further histologic classification into non-invasive ductal or lobular carcinoma (DCIS) and invasive carcinoma (ILC or IDC) underscores its heterogeneity. The ubiquitin-proteasome system plays a crucial role in breast cancer, with inhibitors targeting the 26S proteasome showing promise in clinical treatment. The Cullin-RING ubiquitin ligases, including CUL3, have direct links to breast cancer. This study focuses on CUL3 as a potential biomarker, leveraging high-throughput sequencing, gene expression profiling, experimental and data analysis tools. Through comprehensive analysis using databases like GEPIA2 and UALCAN, as well as TCGA datasets, CUL3's expression and its association with prognostic values were assessed. Additionally, the impact of CUL3 overexpression was explored in MCF-7 and MDA-MB-231 breast cancer cell lines, revealing distinct differences in molecular and phenotypic characteristics. We further profiled its expression and localization in breast cancer tissues identifying prominent differences between luminal A and TNBC tumors. Conclusively, CUL3 was found to be associated with cell cycle progression, and DNA damage response, exhibiting diverse roles depending on the tumor's molecular type. It exhibits a tendency to act as an oncogene in triple-negative tumors and as a tumor suppressor in luminal A types, suggesting a potential significance in breast cancer progression and therapeutic directions.

Mismatch repair protein deficiency in triple-negative breast carcinomas.

BACKGROUND: Breast cancer, particularly triple-negative breast cancer (TNBC), poses a significant global health burden. Chemotherapy was the mainstay treatment for TNBC patients until immunotherapy was introduced. Studies indicate a noteworthy prevalence (0.2% to 18.6%) of mismatch repair protein (MMRP) deficiency in TNBC, with recent research highlighting the potential of immunotherapy for MMRP-deficient metastatic breast cancer. This study aims to identify MMRP deficiency in TNBC patients using immunohistochemistry. METHODS: A retrospective cohort study design was used and included TNBC patients treated between 2015 and 2021 at King Hussein Cancer Center. Immunohistochemistry was conducted to assess MMRP expression. RESULTS: Among 152 patients, 14 (9.2%) exhibited deficient MMR (dMMR). Loss of PMS2 expression was observed in 13 patients, 5 of whom showed loss of MLH1 expression. Loss of MSH6 and MSH2 expression was observed in one patient. The median follow-up duration was 44 (3-102) months. Despite the higher survival rate (80.8%, 5 years) of dMMR patients than of proficient MMR patients (62.3%), overall survival did not significantly differ between the two groups. CONCLUSION: Approximately 9% of TNBC patients exhibit dMMR. dMMR could be used to predict outcomes and identify patients with TNBC who may benefit from immunotherapy.

BRCA1 Intragenic Duplication Combined with a Likely Pathogenic TP53 Variant in a Patient with Triple-Negative Breast Cancer: Clinical Risk and Management.

For patients with hereditary breast and ovarian cancer, the probability of carrying two pathogenic variants (PVs) in dominant cancer-predisposing genes is rare. Using targeted next-generation sequencing (NGS), we investigated a 49-year-old Caucasian woman who developed a highly aggressive breast tumor. Our analyses identified an intragenic germline heterozygous duplication in BRCA1 with an additional likely PV in the TP53 gene. The BRCA1 variant was confirmed by multiplex ligation probe amplification (MLPA), and genomic breakpoints were characterized at the nucleotide level (c.135-2578_442-1104dup). mRNA extracted from lymphocytes was amplified by RT-PCR and then Sanger sequenced, revealing a tandem duplication r.135_441dup; p.(Gln148Ilefs*20). This duplication results in the synthesis of a truncated and, most likely, nonfunctional protein. Following functional studies, the TP53 exon 5 c.472C > T; p.(Arg158Cys) missense variant was classified as likely pathogenic by the Li-Fraumeni Syndrome (LFS) working group. This type of unexpected association will be increasingly identified in the future, with the switch from targeted BRCA sequencing to hereditary breast and ovarian cancer (HBOC) panel sequencing, raising the question of how these patients should be managed. It is therefore important to record and investigate these rare double-heterozygous genotypes.

The potential mechanism of HIF-1α and CD147 in the development of triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis, and the outcomes of common therapy were not favorable. METHODS: The samples of 84 patients with TNBC and 40 patients with breast fibroadenoma were collected in the pathology department specimen library of our hospital. The prognosis of patients was obtained through outpatient follow-up information, telephone and WeChat contacts, and medical records. The mRNA expression was analyzed using bioinformation and quantitative real-time polymerase chain reaction (qPCR). The protein expression was determined by hematoxylin-eosin staining and immunohistochemical staining. The results of survival analysis were visualized using Kaplan-Meier curves. RESULTS: The immunohistochemical staining showed that hypoxia-inducible factor-1alpha (HIF-1α) was mainly distributed in the nucleus and cytoplasm, while CD147 is mainly distributed in cell membrane and cytoplasm. The qPCR results exhibited that the expression level of HIF-1α and CD147 in TNBC tissue was significantly higher than that in breast fibroadenoma tissue. The expression of HIF-1α was related to the histological grade and lymph node metastasis in TNBC, and the expression of CD147 was related to Ki-67, histological grade and lymph node metastasis. There was a positive relationship between the expression of CD147 and HIF-1α. The upregulated expression of CD147 was closely related to the poor prognosis of OS in TNBC. CONCLUSION: CD147 could be a biomarker for the prognosis of TNBC and closely related to the expression of HIF-1α.

Study on the mechanism of heterogeneous tumor-associated macrophages in three subtypes of breast cancer through the integration of single-cell RNA sequencing and in vitro experiments.

BACKGROUND: Tumor-associated macrophages (TAM) exert a significant influence on the progression and heterogeneity of various subtypes of breast cancer (BRCA). However, the roles of heterogeneous TAM within BRCA subtypes remain unclear. Therefore, this study sought to elucidate the role of TAM across the following three BRCA subtypes: triple-negative breast cancer, luminal, and HER2. MATERIALS AND METHODS: This investigation aimed to delineate the variations in marker genes, drug sensitivity, and cellular communication among TAM across the three BRCA subtypes. We identified specific ligand-receptor (L-R) pairs and downstream mechanisms regulated by VEGFA-VEGFR1, SPP1-CD44, and SPP1-ITGB1 L-R pairs. Experimental verification of these pairs was conducted by co-culturing macrophages with three subtypes of BRCA cells. RESULTS: Our findings reveal the heterogeneity of macrophages within the three BRCA subtypes, evidenced by variations in marker gene expression, composition, and functional characteristics. Notably, heterogeneous TAM were found to promote invasive migration and epithelial-mesenchymal transition (EMT) in MDA-MB-231, MCF-7, and SKBR3 cells, activating NF-κB pathway via P38 MAPK, TGF-ß1, and AKT, respectively, through distinct VEGFA-VEGFR1, SPP1-CD44, and SPP1-ITGB1 L-R pairs. Inhibition of these specific L-R pairs effectively reversed EMT, migration, and invasion of each cancer cells. Furthermore, we observed a correlation between ligand gene expression and TAM sensitivity to anticancer drugs, suggesting a potential strategy for optimizing personalized treatment guidance. CONCLUSION: Our study highlights the capacity of heterogeneous TAM to modulate biological functions via distinct pathways mediated by specific L-R pairs within diverse BRCA subtypes. This study might provide insights into precision immunotherapy of different subtypes of BRCA.

Increased expression of REG3A promotes tumorigenic behavior in triple negative breast cancer cells.

BACKGROUND: Identifying new targets in triple negative breast cancer (TNBC) remains critical. REG3A (regenerating islet-derived protein 3 A), a calcium-dependent lectin protein, was thoroughly investigated for its expression and functions in breast cancer. METHODS: Bioinformatics and local tissue analyses were employed to identify REG3A expression in breast cancer. Genetic techniques were employed to modify REG3A expression, and the resulting effects on the behaviors of breast cancer cells were examined. Subcutaneous xenograft models were established to investigate the involvement of REG3A in the in vivo growth of breast cancer cells. RESULTS: Analysis of the TCGA database uncovered increased REG3A levels in human breast cancer tissues. Additionally, REG3A mRNA and protein levels were elevated in TNBC tissues of locally treated patients, contrasting with low expression in adjacent normal tissues. In primary human TNBC cells REG3A shRNA notably hindered cell proliferation, migration, and invasion while triggering caspase-mediated apoptosis. Similarly, employing CRISPR-sgRNA for REG3A knockout showed significant anti-TNBC cell activity. Conversely, REG3A overexpression bolstered cell proliferation and migration. REG3A proved crucial for activating the Akt-mTOR cascade, as evidenced by decreased Akt-S6K1 phosphorylation upon REG3A silencing or knockout, which was reversed by REG3A overexpression. A constitutively active mutant S473D Akt1 (caAkt1) restored Akt-mTOR activation and counteracted the proliferation inhibition and apoptosis induced by REG3A knockdown in breast cancer cells. Crucially, REG3A played a key role in maintaining mTOR complex integrity. Bioinformatics identified zinc finger protein 680 (ZNF680) as a potential REG3A transcription factor. Knocking down or knocking out ZNF680 reduced REG3A expression, while its overexpression increased it in primary breast cancer cells. Additionally, enhanced binding between ZNF680 protein and the REG3A promoter was observed in breast cancer tissues and cells. In vivo, REG3A shRNA significantly inhibited primary TNBC cell xenograft growth. In REG3A-silenced xenograft tissues, reduced REG3A levels, Akt-mTOR inhibition, and activated apoptosis were evident. CONCLUSION: ZNF680-caused REG3A overexpression drives tumorigenesis in breast cancer possibly by stimulating Akt-mTOR activation, emerging as a promising and innovative cancer target.

TFAP2A downregulation mediates tumor-suppressive effect of miR-8072 in triple-negative breast cancer via inhibiting SNAI1 transcription.

BACKGROUND: Triple-negative breast cancer (TNBC) represents a highly aggressive subset of breast malignancies characterized by its challenging clinical management and unfavorable prognosis. While TFAP2A, a member of the AP-2 transcription factor family, has been implicated in maintaining the basal phenotype of breast cancer, its precise regulatory role in TNBC remains undefined. METHODS: In vitro assessments of TNBC cell growth and migratory potential were conducted using MTS, colony formation, and EdU assays. Quantitative PCR was employed to analyze mRNA expression levels, while Western blot was utilized to evaluate protein expression and phosphorylation status of AKT and ERK. The post-transcriptional regulation of TFAP2A by miR-8072 and the transcriptional activation of SNAI1 by TFAP2A were investigated through luciferase reporter assays. A xenograft mouse model was employed to assess the in vivo growth capacity of TNBC cells. RESULTS: Selective silencing of TFAP2A significantly impeded the proliferation and migration of TNBC cells, with elevated TFAP2A expression observed in breast cancer tissues. Notably, TNBC patients exhibiting heightened TFAP2A levels experienced abbreviated overall survival. Mechanistically, TFAP2A was identified as a transcriptional activator of SNAI1, a crucial regulator of epithelial-mesenchymal transition (EMT) and cellular proliferation, thereby augmenting the oncogenic properties of TFAP2A in TNBC. Moreover, miR-8072 was unveiled as a negative regulator of TFAP2A, exerting potent inhibitory effects on TNBC cell growth and migration. Importantly, the tumor-suppressive actions mediated by the miR-8072/TFAP2A axis were intricately associated with the attenuation of AKT/ERK signaling cascades and the blockade of EMT processes. CONCLUSIONS: Our findings unravel the role and underlying molecular mechanism of TFAP2A in driving tumorigenesis of TNBC. Targeting the TFAP2A/SNAI1 pathway and utilizing miR-8072 as a suppressor represent promising therapeutic strategies for treating TNBC.

[A recombinant adeno-associated virus expressing secretory TGF-ß type â ¡ receptor inhibits triple-negative murine breast cancer 4T1 cell proliferation and lung metastasis in mice].

OBJECTIVE: To investigate the effects of an adeno-associated virus (AAV2) vector expressing secretory transforming growth factor-ß (TGF-ß) type Ⅱ receptor (sTßRⅡ) extracellular domain-IgG2a Fc fusion protein (sTßRⅡ-Fc) on proliferation and migration of triple-negative murine breast cancer 4T1 cells in mice. METHODS: The pAAV-sTßRⅡ-Fc vector expressing sTßRⅡ-Fc fusion protein constructed by molecular cloning, the capsid protein-expressing vector pAAV2 and the helper vector were co-transfected into HEK 293T cells to prepare the recombinant AAV2-sTßRⅡ virus, which was purified by density gradient centrifugation with iodixanol. Western blotting was used to examine the effects of AAV-sTßRⅡ virus on Smad2/3 phosphorylation in 4T1 cells and on expression levels of E-cadherin, vimentin and p-Smad2/3 in 4T1 cell xenografts in mice. BALB/c mice bearing subcutaneous xenografts of luciferase-expressing 4T1 cells received intravenous injections of AAV-sTßRⅡ virus, AAV-GFP virus or PBS (n=6) through the tail vein, and the proliferation and migration of 4T1 cells were analyzed with in vivo imaging. Ki67 expression in the tumor tissues and sTßRⅡ protein expressions in mouse livers were detected with immunohistochemistry and immunofluorescence staining, and tumor metastases in the vital organs were examined with HE staining. RESULTS: The recombinant pAAV-sTßRⅡ-Fc vector successfully expressed sTßRⅡ in HEK 293T cells. Infection with AAV2-sTßRⅡ virus significantly reduced TGF-ß1-induced Smad2/3 phosphorylation in 4T1 cells and effectively inhibited proliferation and lung metastasis of 4T1 xenografts in mice (P<0.05). In the tumor-bearing mice, intravenous injection of AAV-sTßRⅡ virus significantly increased E-cadherin expression, reduced vimentin and Ki67 protein expressions and Smad2/3 phosphorylation level in the tumor tissues (P<0.05 or 0.01), and induced liver-specific sTßRⅡ expression without causing body weight loss or heart, liver, spleen or kidney pathologies. CONCLUSION: The recombinant AVV2 vector encoding sTßRⅡ extracellular domain is capable of blocking the TGF-ß signaling pathway to inhibit the proliferation and lung metastasis of 4T1 cells in mice.

Mechanisms underlying neutrophils adhesion to triple-negative breast cancer cells via CD11b-ICAM1 in promoting breast cancer progression.

BACKGROUND: Triple-negative breast cancer (TNBC) is recognized as the most aggressive and immunologically infiltrated subtype of breast cancer. A high circulating neutrophil-to-lymphocyte ratio (NLR) is strongly linked to a poor prognosis among patients with breast cancer, emphasizing the critical role of neutrophils. Although the involvement of neutrophils in tumor metastasis is well documented, their interactions with primary tumors and tumor cells are not yet fully understood. METHODS: Clinical data were analyzed to investigate the role of neutrophils in breast cancer. In vivo mouse model and in vitro co-culture system were used for mechanism researches. Blocking experiments were further performed to identify therapeutic agents against TNBC. RESULTS: TNBC cells secreted GM-CSF to sustain the survival of mature neutrophils and upregulated CD11b expression. Through CD11b, neutrophils specifically binded to ICAM1 on TNBC cells, facilitating adhesion. Transcriptomic sequencing combined with human and murine functional experiments revealed that neutrophils, through direct CD11b-ICAM1 interactions, activated the MAPK signaling pathway in TNBC cells, thereby enhancing tumor cell invasion and migration. Atorvastatin effectively inhibited ICAM1 expression in tumor cells, and tumor cells with ICAM1 knockout or treated with atorvastatin were unresponsive to neutrophil activation. The MAPK pathway and MMP9 expression were significantly inhibited in the tumor tissues of TNBC patients treated with atorvastatin. CONCLUSIONS: Targeting CD11b-ICAM1 with atorvastatin represented a potential clinical approach to reduce the malignant characteristics of TNBC.

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment. METHODS: Gene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays. RESULTS: BUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade. CONCLUSIONS: BUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

YY1 mediated DCUN1D5 transcriptional activation promotes triple-negative breast cancer progression by targeting FN1/PI3K/AKT pathway.

Triple-negative breast cancer (TNBC) is more aggressive and has a higher metastasis rate compared with other subtypes of breast cancer. Due to the lack of drug-targetable receptors, chemotherapy is now the only available systemic treatment for TNBC. However, some patients might still develop drug resistance and have poor prognosis. Therefore, novel molecular biomarkers and new treatment targets are urgently needed for patients with TNBC. To provide molecular insights into TNBC progression, we investigated the function and the underlying mechanism of Defective in cullin neddylation 1 domain containing 5 (DCUN1D5) in the regulation of TNBC. By TCGA dataset and surgical specimens with immunohistochemical (IHC) staining method, DCUN1D5 was identified to be significantly upregulated in TNBC tumor tissues and negatively associated with prognosis. A series of in vitro and in vivo experiments were performed to confirm the oncogenic role of DCUN1D5 in TNBC. Overexpression of FN1 or PI3K/AKT activator IGF-1 could restore the proliferative and invasive ability induced by DCUN1D5 knockdown and DCUN1D5 could act as a novel transcriptional target of transcription factor Yin Yang 1 (YY1). In conclusion, YY1-enhanced DCUN1D5 expression could promote TNBC progression by FN1/PI3K/AKT pathway and DCUN1D5 might be a potential prognostic biomarker and therapeutic target for TNBC treatment.

Exosomal circSIPA1L3-mediated intercellular communication contributes to glucose metabolic reprogramming and progression of triple negative breast cancer.

BACKGROUND: Breast cancer is the most common malignant tumor, and metastasis remains the major cause of poor prognosis. Glucose metabolic reprogramming is one of the prominent hallmarks in cancer, providing nutrients and energy to support dramatically elevated tumor growth and metastasis. Nevertheless, the potential mechanistic links between glycolysis and breast cancer progression have not been thoroughly elucidated. METHODS: RNA-seq analysis was used to identify glucose metabolism-related circRNAs. The expression of circSIPA1L3 in breast cancer tissues and serum was examined by qRT-PCR, and further assessed its diagnostic value. We also evaluated the prognostic potential of circSIPA1L3 by analyzing a cohort of 238 breast cancer patients. Gain- and loss-of-function experiments, transcriptomic analysis, and molecular biology experiments were conducted to explore the biological function and regulatory mechanism of circSIPA1L3. RESULTS: Using RNA-seq analysis, circSIPA1L3 was identified as the critical mediator responsible for metabolic adaption upon energy stress. Gain- and loss-of-function experiments revealed that circSIPA1L3 exerted a stimulative effect on breast cancer progression and glycolysis, which could also be transported by exosomes and facilitated malignant behaviors among breast cancer cells. Significantly, the elevated lactate secretion caused by circSIPA1L3-mediated glycolysis enhancement promoted the recruitment of tumor associated macrophage and their tumor-promoting roles. Mechanistically, EIF4A3 induced the cyclization and cytoplasmic export of circSIPA1L3, which inhibited ubiquitin-mediated IGF2BP3 degradation through enhancing the UPS7-IGF2BP3 interaction. Furthermore, circSIPA1L3 increased mRNA stability of the lactate export carrier SLC16A1 and the glucose intake enhancer RAB11A through either strengthening their interaction with IGF2BP3 or sponging miR-665, leading to enhanced glycolytic metabolism. Clinically, elevated circSIPA1L3 expression indicated unfavorable prognosis base on the cohort of 238 breast cancer patients. Moreover, circSIPA1L3 was highly expressed in the serum of breast cancer patients and exhibited high diagnostic value for breast cancer patients. CONCLUSIONS: Our study highlights the oncogenic role of circSIPA1L3 through mediating glucose metabolism, which might serve as a promising diagnostic and prognostic biomarker and potential therapeutic target for breast cancer.

Genome-wide in vivo CRISPR screen identifies TGFß3 as actionable biomarker of palbociclib resistance in triple negative breast cancer.

Triple negative breast cancer (TNBC) remains exceptionally challenging to treat. While CDK4/6 inhibitors have revolutionized HR + breast cancer therapy, there is limited understanding of their efficacy in TNBC and meaningful predictors of response and resistance to these drugs remain scarce. We conducted an in vivo genome-wide CRISPR screen using palbociclib as a selection pressure in TNBC. Hits were prioritized using microarray data from a large panel of breast cancer cell lines to identify top palbociclib sensitizers. Our study defines TGFß3 as an actionable determinant of palbociclib sensitivity that potentiates its anti-tumor effects. Mechanistically, we show that chronic palbociclib exposure depletes p21 levels, contributing to acquired resistance, and that TGFß3 treatment can overcome this. This study defines TGFß3 as an actionable biomarker that can be used to improve patient stratification for palbociclib treatment and exploits the synergistic interaction between CDK4/6 and TGFß3 to propose a new combinatorial treatment for TNBC.

Low BCL-xL expression in triple-negative breast cancer cells favors chemotherapy efficacy, and this effect is limited by cancer-associated fibroblasts.

Triple negative breast cancers (TNBC) present a poor prognosis primarily due to their resistance to chemotherapy. This resistance is known to be associated with elevated expression of certain anti-apoptotic members within the proteins of the BCL-2 family (namely BCL-xL, MCL-1 and BCL-2). These regulate cell death by inhibiting pro-apoptotic protein activation through binding and sequestration and they can be selectively antagonized by BH3 mimetics. Yet the individual influences of BCL-xL, MCL-1, and BCL-2 on the sensitivity of TNBC cells to chemotherapy, and their regulation by cancer-associated fibroblasts (CAFs), major components of the tumor stroma and key contributors to therapy resistance remain to be delineated. Using gene editing or BH3 mimetics to inhibit anti-apoptotic BCL-2 family proteins in TNBC line MDA-MB-231, we show that BCL-xL and MCL-1 promote cancer cell survival through compensatory mechanisms. This cell line shows limited sensitivity to chemotherapy, in line with the clinical resistance observed in TNBC patients. We elucidate that BCL-xL plays a pivotal role in therapy response, as its depletion or pharmacological inhibition heightened chemotherapy effectiveness. Moreover, BCL-xL expression is associated with chemotherapy resistance in patient-derived tumoroids where its pharmacological inhibition enhances ex vivo response to chemotherapy. In a co-culture model of cancer cells and CAFs, we observe that even in a context where BCL-xL reduced expression renders cancer cells more susceptible to chemotherapy, those in contact with CAFs display reduced sensitivity to chemotherapy. Thus CAFs exert a profound pro-survival effect in breast cancer cells, even in a setting highly favoring cell death through combined chemotherapy and absence of the main actor of chemoresistance, BCL-xL.