Prognosis In Triple-negative Breast Cancer Patients Research Articles

Quantifying spatial CXCL9 distribution with image analysis predicts improved prognosis of triple-negative breast cancer.

Background: The C-X-C motif chemokine ligand 9 (CXCL9) plays a pivotal role in tumor immunity by recruiting and activating immune cells. However, the relationship between CXCL9 expression and prognosis in triple-negative breast cancer (TNBC) is unclear. Methods: We investigated CXCL9 mRNA expression, clinicopathological features, and prognosis in TNBC patients. We also used computational image analysis to quantify and assess the distribution of CXCL9 protein in the tumor core (TC) and invasive margin (IM). Results: CXCL9 mRNA expression was significantly higher in TNBC tumors compared to normal tissue (p < 0.001) and was associated with smaller tumors (p = 0.022) and earlier stages (p = 0.033). High CXCL9 mRNA expression was correlated with improved overall survival (OS) in three independent cohorts (all p < 0.05). In a separate analysis, low CXCL9 protein expression was associated with increased lymph node metastasis (p = 0.018 and p = 0.036). High CXCL9 protein expression in the TC, IM, or both was associated with prolonged OS (all p < 0.001). Conclusion: High CXCL9 expression, at both the mRNA and protein levels, is associated with improved prognosis in TNBC patients. CXCL9 expression in the TC and/or IM may be an independent prognostic factor.

Effect of transcription factor WT1 on triple-negative breast cancer metastasis through PFKFB4-mediated glycolysis.

e13145 Background: Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer with a high probability of metastasis as well as a lack of specific targets and targeted therapeutics. Preliminary study suggested that Wilms' tumor gene 1 (WT1) is highly expressed in breast cancer patients with poor prognosis and may promote TNBC metastasis, but the underlying mechanism remains poorly defined. Methods: WT1 expression was evaluated by immunohistochemistry (IHC) in breast cancer patients. Kaplan–Meier survival analysis was performed to assess the prognostic significance of WT1 expression. WT1 was silenced in MDA-MB-231 and BT549 cells or overexpressed in HCC1806 cells. qRT-PCR and Western blot were used to detect the WT1 expression in tissues and cells. Wound healing assays, transwell assays and 3D spheroid assays were used to examine the migration and invasion abilities in TNBC cells. The lung metastasis model of mice was used to evaluate metastasis of TNBC in vivo. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) and transcriptome sequencing (RNA-seq) were performed to find PFKFB4, a downstream target gene for WT1. ChIP-PCR and dual-luciferase reporter assays were used to explore the transcriptional regulation of PFKFB4 by WT1. Seahorse XF glycolysis stress assay, glucose uptake assay, and lactate production assay were used to investigate the role of WT1 in regulating glycolysis metabolites. Results: WT1 was highly expressed in TNBC and correlated to poor prognosis in TNBC patients. Functional assays revealed that WT1 promoted TNBC cell metastasis in vitro and in vivo. Our mechanistic investigations demonstrated that WT1 promoted TNBC cells migration and invasion by transcriptionally activating the expression of PFKFB4.This action leaded to increased glycolytic capacity, glucose uptake, and lactate production in cancer cells, therefore promoting metastasis of TNBC. Clinically, the combined expression of WT1 and PFKFB4 provides a reliable predictive biomarker for the prognosis of TNBC patients. Conclusions: Our findings reveal a molecular mechanism of WT1 promoting TNBC metastasis, which provides new targets for the precision treatment of TNBC and new perspectives for the development of targeted metabolic anticancer drugs.

Abstract PO3-25-07: Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics

Abstract Background Tertiary lymphoid structures (TLS) are ectopic lymphoid organs playing a role in adaptive antitumor immune response. They were shown to be associated with favorable outcome and appears to be a promising biomarker for response to immune checkpoint inhibitors (ICI). Yet, there is no consensus for their detection and quantification. Here we aimed to derive a specific TLS signature using high resolution spatial transcriptomics data from a large series of triple negative breast cancer (TNBC) samples and to assess its clinical relevance including response to ICI. Methods Spatial transcriptomics (ST) was performed on a series of 94 early-stage TNBC surgical samples with detailed clinicopathological and outcome data. TLS and tumor infiltrating lymphocytes (TILs) were scored using hematoxylin and eosin and double immunohistochemistry (CD3/CD20) stained slides by a dedicated breast pathologist. Regression based on morphological annotations and deconvolution methods estimated the fraction of gene expression related to TLS in each ST spot allowing to derive specific TLS signature. Its prognostic and predictive value of response to ICI was assessed using publicly available breast cancer (METABRIC, SCAN-B, and I-SPY2) and other tumor type datasets treated with ICI (Bareche et al. Ann Oncol, 2022) from bulk gene expression data. Results Deconvolution of the TLS compartments using the ST gene expression data showed that TLS are enriched in all B cell subsets, CD4+ (central) memory and naïve T cells, as well as mast cells. A functional analysis showed an enrichment of ‘vascular endothelial cell proliferation’, ‘mitotic G2/M transition checkpoint’, ‘V/D/J recombination’ and ‘regulation of cell chemotaxis to fibroblast growth factor’ suggesting that TLS aggregates are associated with angiogenesis, immune cell proliferation, adaptative humoral response and tissue remodeling. A comparison of gene expression data between TLS and non-aggregated TILs led to the development of a specific TLS gene signature comprising B and T cell-specific genes, immunoglobulin genes as well as genes associated with TLS initiation. Its projection on tumor slides overlapped with the regions annotated as TLS by the pathologist, demonstrating its specificity for TLS detection. As expected, the highest levels of the TLS signature were observed in the immunomodulatory TNBC molecular subtype and fully inflamed tumors, whereas the lowest levels were observed in the mesenchymal molecular subtype and immune desert tumors (p&amp;lt; 0.05). Of note, high levels of the TLS signature were associated with better prognosis in TNBC patients from METABRIC (p=6.5 10−5) and SCAN-B datasets (p=1.6 10−3) as well as with higher pathological complete response rates in all breast cancer patients treated with neoadjuvant pembrolizumab in the I-SPY2 study (p=0.026). Similar results were found in other tumor types treated with immunotherapy in the metastatic setting including metastatic melanoma, pancreatic and bladder cancers (PFS, p=6 10−6). Of interest, the TLS signature outperformed the predictive value of other immune-related signatures including TILs suggesting a key role of TLS in obtaining a sustainable, adaptive antitumor immune response in the vicinity of the tumor area. Conclusion By leveraging the potential of spatial transcriptomics, we have successfully developed a unique TLS gene signature that demonstrates a strong correlation with the response to ICI in breast cancer and various other tumor types. This TLS gene signature outperforms other immune biomarkers, underscoring the pivotal role of TLS in eliciting a robust antitumor immune response. The universality of this TLS signature makes it a potent biomarker capable of identifying patients who would benefit from immunotherapy, pending further validation. Citation Format: Xiaoxiao Wang, David Venet, Frédéric Lifrange, Denis Larsimont, Mattia Rediti, Linnea Stenbeck, Floriane Dupont, Ghizlane Rouas, Andrea Joaquin Garcia, David Gacquer, Ligia Craciun, Laurence Buisseret, Nayanika Bhalla, Yuvarani Masarapu, Kim Thrane, Eva Gracia Villacampa, Lovisa Franzén, Sami Saarenpää, Linda Kvastad, Joakim Lundeberg, Françoise Rothé, Christos Sotiriou. Characterization of tertiary lymphoid structures and its association with response to immune checkpoint inhibitors in triple negative breast cancer using spatial transcriptomics [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-25-07.

Abstract PO3-13-06: TILs are an independent prognostic factor in early-stage TNBC patients achieving pathologic complete response after neoadjuvant chemotherapy

Abstract Introduction: In patients with triple negative breast cancer (TNBC) the achievement of pathological complete response (pCR) after neoadjuvant chemotherapy (NACT) is associated with better long-term clinical outcomes. However, the attainment of pCR does not guarantee cure, since ~ 10% of patients undergo disease relapse. Higher levels of tumor infiltrating lymphocytes (TILs) are associated with better prognosis in TNBC patients undergoing upfront surgery and in patients with residual disease after NACT. However, the prognostic role of TILs among TNBC patients undergoing pCR during NACT has not been investigated so far. Methods: We included 134 TNBC patients (ER cutoff&amp;lt; 10%) who achieved pCR after NACT. These cases were identified from a cohort of 348 TNBC patients who received NACT at Istituto Oncologico Veneto (Padova) and Istituto Nazionale Tumori (Milano) from April 2004 to April 2021. TILs were quantified in pre-NACT tumor biopsy according to International guidelines. We assessed the association between TILs and relapse-free survival (RFS) or overall survival (OS). Results: Main patients characteristics were: median age 48 years; ductal histotype 92.8%; histologic grade 3 95.2%; stage I 9.7%, stage II 71.6%, stage III 18.7%. Almost all patients received anthracyclines (98.5%) and taxanes (99.3%) as part of NACT for a median number of 4 cycles each; 55.2% of patients received carboplatin. At a median FU of 4.6 years, the 5-yr RFS and OS rates were 90.4% and 94.4%, respectively. As a continuous variable (1% increment), TILs were independently associated with better RFS and OS after adjusting for patient age and tumor stage: HR 0.90 (95%CI 0.83-0.98), p=0.015 and HR 0.89 (95%CI 0.80-1.00), p=0.041, respectively. The best prognostic TILs cut-off in this cohort was 20%. Patients with TILs&amp;gt;20% showed a 100% 5-yr RFS rate (0/63 events) vs 82.6% (12/71 events) for patients with TILs&amp;lt; 20% (log-rank p&amp;lt; 0.001). OS rates at 5 years were 100% and 90.1% for high vs low TILs (log-rank p=0.007). Conclusions: This is the first demonstration of an independent prognostic role of baseline TILs in TNBC patients achieving pCR after NACT. Baseline TILs may discriminate patients who are cured after achieving pCR from patients at risk of disease relapse despite the achievement of pCR. These latter may be candidate to treatment escalation in the context of clinical trials. Our work provides relevant data in the ongoing debate on the role of adjuvant therapy (primarily pembrolizumab) following pCR. Citation Format: Maria Vittoria Dieci, Claudio Vernieri, Davide Massa, Lorenzo Nicolè, Gaia Griguolo, Federica Miglietta, Andrea Vingiani, Riccardo Lobefaro, Fabio Girardi, Grazia Vernaci, Tommaso Giarratano, Carlo Alberto Giorgi, Giancarlo Pruneri, Matteo Fassan, Filippo De Braud, Valentina Guarneri. TILs are an independent prognostic factor in early-stage TNBC patients achieving pathologic complete response after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-13-06.

PYCR3 modulates mtDNA copy number to drive proliferation and doxorubicin resistance in triple-negative breast cancer

Triple-negative breast cancer (TNBC) poses significant challenges in treatment due to its aggressive nature and limited therapeutic targets. Understanding the underlying molecular mechanisms driving TNBC progression and chemotherapy resistance is imperative for developing effective therapeutic strategies. Thus, in this study, we aimed to elucidate the role of pyrroline-5-carboxylate reductase 3 (PYCR3) in TNBC pathogenesis and therapeutic response. We observed that PYCR3 is significantly upregulated in TNBC specimens compared to normal breast tissues, correlating with a poorer prognosis in TNBC patients. Knockdown of PYCR3 not only suppresses TNBC cell proliferation but also reverses acquired resistance of TNBC cells to doxorubicin, a commonly used chemotherapeutic agent. Mechanistically, we identified the mitochondrial localization of PYCR3 in TNBC cells and demonstrated its impact on TNBC cell proliferation and sensitivity to doxorubicin through the regulation of mtDNA copy number and mitochondrial respiration. Importantly, Selective reduction of mtDNA copy number using the mtDNA replication inhibitor 2′, 3′-dideoxycytidine effectively recapitulates the phenotypic effects observed in PYCR3 knockout, resulting in decreased TNBC cell proliferation and the reversal of doxorubicin resistance through apoptosis induction. Thus, our study underscores the clinical relevance of PYCR3 and highlight its potential as a therapeutic target in TNBC management. By elucidating the functional significance of PYCR3 in TNBC, our findings contribute to a deeper understanding of TNBC biology and provide a foundation for developing novel therapeutic strategies aimed at improving patient outcomes.

PDZK1 suppresses TNBC development and sensitizes TNBC cells to erlotinib via the EGFR pathway

Epidermal growth factor receptor (EGFR)-targeted drugs (erlotinib, etc.) are used to treat multiple types of tumours. EGFR is highly expressed in most triple-negative breast cancer (TNBC) patients. However, only a small proportion of TNBC patients benefit from EGFR-targeted drugs in clinical trials, and the resistance mechanism is unclear. Here, we found that PDZ domain containing 1 (PDZK1) is downregulated in erlotinib-resistant TNBC cells, suggesting that PDZK1 downregulation is related to erlotinib resistance in TNBC. PDZK1 binds to EGFR. Through this interaction, PDZK1 promotes EGFR degradation by enhancing the binding of EGFR to c-Cbl and inhibits EGFR phosphorylation by hindering EGFR dimerisation. We also found that PDZK1 is specifically downregulated in TNBC tissues and correlated with a poor prognosis in TNBC patients. In vitro and in vivo functional assays showed that PDZK1 suppressed TNBC development. Restoration of EGFR expression or kinase inhibitor treatment reversed the degree of cell malignancy induced by PDZK1 overexpression or knockdown, respectively. PDZK1 overexpression sensitised TNBC cells to erlotinib both in vitro and in vivo. In conclusion, PDZK1 is a significant prognostic factor for TNBC and a potential molecular therapeutic target for reversing erlotinib resistance in TNBC cells.

Targeting CXCR4/CXCL12 axis via [177Lu]Lu-DOTAGA.(SA.FAPi)2 with CXCR4 antagonist in triple-negative breast cancer

PurposeRadiopharmaceutical therapies targeting fibroblast activation protein (FAP) have shown promising efficacy against many tumor types. But radiopharmaceuticals alone in most cases are insufficient to completely eradicate tumor cells, which can partially be attributed to the protective interplay between tumor cells and cancer-associated fibroblasts (CAFs). The C-X-C chemokine receptor type 4/C-X-C motif chemokine 12 (CXCR4/CXCL12) interaction plays an important role in orchestrating tumor cells and CAFs. We hereby investigated the feasibility and efficacy of [177Lu]Lu-DOTAGA.(SA.FAPi)2, a FAP-targeting radiopharmaceutical, in combination with AMD3100, a CXCR4 antagonist, in a preclinical murine model of triple-negative breast cancer (TNBC).MethodsPublic database was first interrogated to reveal the correlation between CAFs’ scores and the prognosis of TNBC patients, as well as the expression levels of FAP and CXCR4 in normal tissues and tumors. In vitro therapeutic efficacy regarding cell proliferation, migration, and colony formation was assessed in BALB/3T3 fibroblasts and 4T1 murine breast cancer cells. In vivo therapeutic efficacy was longitudinally monitored using serial 18F-FDG, [18F]AlF-NOTA-FAPI-04, and [68Ga]Ga-DOTA-Pentixafor PET/CT scans and validated using tumor sections through immunohistochemical staining of Ki-67, α-SMA, CXCR4, and CXCL12. Intratumoral abundance of myeloid-derived suppressive cells (MDSCs) was analyzed using flow cytometry in accordance with the PET/CT schedules. Treatment toxicity was evaluated by examining major organs including heart, lung, liver, kidney, and spleen.ResultsCAFs’ scores negatively correlated with the survival of TNBC patients (p < 0.05). The expression of CXCR4 and FAP was both significantly higher in tumors than in normal tissues. The combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 significantly suppressed cell proliferation, migration, and colony formation in cell culture, and exhibited synergistic effects in 4T1 tumor models along with a decreased number of MDSCs. PET/CT imaging revealed lowest tumor accumulation of 18F-FDG and [18F]AlF-NOTA-FAPI-04 on day 13 and day 14 after treatment started, both of which gradually increased at later time points. A similar trend was observed in the IHC staining of Ki-67, α-SMA, and CXCL12.ConclusionThe combination of [177Lu]Lu-DOTAGA.(SA.FAPi)2 and AMD3100 is a feasible treatment against TNBC with minimal toxicity in main organs.

PTGER3 knockdown inhibits the vulnerability of triple-negative breast cancer to ferroptosis.

Prostaglandin E receptor 3 (PTGER3) is involved in a variety of biological processes in the human body and is closely associated with the development and progression of a variety of cancer types. However, the role of PTGER3 in triple-negative breast cancer (TNBC) remains unclear. In the present study, low PTGER3 expression was found to be associated with poor prognosis in TNBC patients. PTGER3 plays a crucial role in regulating TNBC cell invasion, migration, and proliferation. Upregulation of PTGER3 weakens the epithelial-mesenchymal phenotype in TNBC and promotes ferroptosis both invitro and invivo by repressing glutathione peroxidase 4 (GPX4) expression. On the other hand, downregulation of PTGER3 inhibits ferroptosis by increasing GPX4 expression and activating the PI3K-AKT pathway. Upregulation of PTGER3 also enhances the sensitivity of TNBC cells to paclitaxel. Overall, this study has elucidated critical pathways in which low PTGER3 expression protects TNBC cells from undergoing ferroptosis, thereby promoting its progression. PTGER3 may thus serve as a novel and promising biomarker and therapeutic target for TNBC.

Abstract 5366: ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression

Abstract The stiffening of the extracellular matrix (ECM), a phenomenon that often promotes metastasis and immunosuppression in many cancers, is a process that is not yet fully understood. Our study reveals that ATR, a molecule primarily recognized for its role in maintaining the genome, plays a crucial role in ECM stiffness-induced immune evasion and metastasis in triple negative breast cancer (TNBC) through DNA repair independent manner. In response to ECM stiffness, ATR activates SUN2, a component of the linker of nucleoskeleton and cytoskeleton (LINC) complex. This activation enhances the interaction of SUN2 with outer nuclear membrane proteins KASH1 and KASH2. The interaction between SUN2 and KASH1/2 facilitates the nuclear localization of β-catenin, which in turn promotes epithelial mesenchymal transition (EMT) and reduces the expression of E-cadherin. E-cadherin is essential for attracting CD103+ immune cells that perform anti-tumor immunity. Interestingly, inhibiting ATR reduces EMT in tumor cells, leading to an increase in CD103+ dendritic cell infiltration while decreasing neutrophil and polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) infiltration. Further, we found that ATR levels are inversely correlated with immunotherapy response rates in patients. The alteration in immune cell composition sensitizes tumors to immunotherapy, thereby suppressing tumor growth and metastasis. In conclusion, the role of ATR in remodeling the immune environment could be crucial for optimizing the clinical use of ATR inhibitors. This makes ATR a promising target for overcoming immunotherapy resistance. We suggest early or prolonged treatment with an ATR inhibitor to potentially reduce metastasis rates and improve the prognosis of TNBC patients. Furthermore, we suggest using ATR levels as a marker to predict response rates and prognosis in TNBC patients. For patients with higher ATR expression levels, combination treatment with ATR inhibitors and immunotherapy may yield better outcomes. Enhanced anti-tumor immunity could increase the chances for patients to eliminate cancer cells and help end breast cancer. Finally, our research suggests the combined treatment of ATR inhibition and immunotherapy for patients without necessarily applying DNA damage stimulation. This approach could potentially reduce toxicity while still benefiting patients. Citation Format: Xinyi Tu, Zhenkun Lou, Robert Mutter. ATR promotes extracellular matrix stiffness mediated cytoskeleton remodeling and immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5366.

Abstract 59: Triple negative breast cancer (TNBC) targeting CAR-T cell constructs with phage display-derived peptides

Abstract Introduction: Triple Negative Breast Cancer (TNBC) poses treatment challenges due to the absence of effective targeted therapies. CAR-T cell therapy, successful in hematological cancers, is now being investigated for solid tumors. Finding new CAR-T targets is vital to overcome TNBC's therapeutic constraints. Methods: In this study, a Biopanning phage display strategy employing a library of random peptides was utilized to identify potential targets for CAR-T cell therapy against TNBC. Through this approach, Heat Shock Protein 60 (HSP60) emerged as a promising candidate due to its binding affinity to the selected phage clones. Subsequent validation of HSP60 surface expression on TNBC cell lines in comparison to normal cells was conducted using flow cytometry. Concurrently, patient survival data were gathered and analyzed to evaluate the clinical significance of HSP60 expression in TNBC. The identified peptides demonstrating high specificity and affinity for HSP60 on the surface of TNBC cells were chosen for the development of a third-generation CAR-T model, incorporating various optimizations and innovations. Results: The Biopanning strategy successfully identified HSP60 as a prominent target for CAR-T cell therapy in TNBC. Flow cytometry analysis confirmed significantly elevated surface expression of HSP60 on TNBC cell lines compared to their normal counterparts, substantiating its potential as an accessible antigen for targeted therapy. Patient survival analysis linked elevated HSP60 levels with poorer prognosis in TNBC, highlighting the protein's clinical relevance. Peptides showing high specificity and binding to TNBC's HSP60 were integrated into a meticulously engineered third-gen CAR-T cell model. This design aimed to boost the CAR-T's precision in targeting and eliminating HSP60-expressing TNBC cells. Iterative optimizations ensured the construct's accuracy, laying the groundwork for a potent, tailored TNBC immunotherapy. Discussion: The discovery of HSP60 as a prime target for CAR-T cell therapy in TNBC is a pivotal advancement. Its distinct presence on TNBC cell surfaces, confirmed via flow cytometry, highlights its suitability for targeted immunotherapy. The correlation between elevated HSP60 levels and poorer prognosis in TNBC patients underscores its value as both a biomarker and a therapeutic focus. The peptides' exceptional specificity and strong binding to HSP60 validate their use in crafting a third-generation CAR-T cell model. Their integration and careful refinements in CAR-T design aim to optimize efficacy while reducing off-target effects, laying a robust foundation for tailored TNBC immunotherapy. Preclinical evaluations are imperative to thoroughly assess the safety and effectiveness of this innovative CAR-T approach. Citation Format: Muhammad G. Khodary, Alehegne W. Yirsaw, Yumna A. Elsobky, Reshma Gurung, Jessie Jaynes, Temesgen Samuel, Maninder Sandey, Deepa Bedi. Triple negative breast cancer (TNBC) targeting CAR-T cell constructs with phage display-derived peptides [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 59.

Abstract 5850: Multiomics analysis unravels tumor metabolic reprogramming and potential resistance mechanism(s) to curative therapy in TNBC

Abstract Drug resistance is the leading cause of treatment failure to completely eliminate viable tumor cells in ~40% of patients with triple negative breast cancer (TNBC). Making a major impact on the treatment and prognosis for TNBC patients requires a better understanding of drug resistance mechanism(s). Combining clinical data with high-throughput metabolomic and proteomic analyses, we probed the molecular basis for chemotherapy resistance in treatment-naive TNBC primary tumors, reflected by pathological complete response (pCR) and non-pCR after neoadjuvant chemotherapy (NAC). Metabolic profiling uncovered that glycolysis and oxidative phosphorylation (OXPHOS) co-exist in TNBC primary tumors compared to benign lesions regardless of pCR status, indicating metabolic reprogramming. However, non-pCR tumors were more enriched in OXPHOS and less in glycolysis relative to pCR tumors, pointing to the potential importance of OXPHOS in drug resistance. Moreover, parallel proteomic profiling of these same TNBC tumors supported the results from metabolic profiling, showing that OXPHOS was indeed highly enriched at the protein level, and may be regulated by SIRT5-mediated sirtuin signaling. In alignment with these findings, non-pCR tumors were abundantly enriched in immune-inhibitory metabolites that may be due to the products of SIRT5-mediated mitochondrial metabolism. Thus, these findings led me to hypothesize that SIRT5 may play an important role in chemoresistance through regulation of mitochondrial metabolism and ROS detoxification. Together, the metabolite resulted from SIRT5-mediated mitochondrial metabolism led to the suppression of immune cell infiltration in non-pCR tumors. Understanding SIRT5-mediated chemoresistance mechanism may uncover a metabolic and therapeutic vulnerability to resistant TNBCs. Citation Format: Zuen Ren, Kiran Kurmi, Robert Morris, Shakchhi Joshi, Eric Zaniewski, Johannes Kreuzer, Gabrielle Elena Gioia, Veerle I. Bossuyt, Brian N. Dontchos, Gary X. Wang, Shinn-Huey S. Chou, Laura M. Spring, Wilhelm Haas, Marcia C. Haigis, Leif W. Ellisen. Multiomics analysis unravels tumor metabolic reprogramming and potential resistance mechanism(s) to curative therapy in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5850.

Development and verification of a novel immunogenic cell death-related signature for predicting the prognosis and immune infiltration in triple-negative breast cancer.

Insufficient understanding of the pathogenesis and tumor immunology of triple-negative breast cancer (TNBC) has limited the development of immunotherapy. The importance of tumor microenvironment (TME) in immunotyping, prognostic assessment and immunotherapy efficacy of cancer has been emphasized, however, potential immunogenic cell death (ICD) related genes function in TME of TNBC has been rarely investigated. To initially explore the role and related mechanisms of ICD in TNBC, especially the role played in the TME of TNBC, and to identify different relevant subtypes based on ICD, and then develop an ICD-related risk score to predict each TNBC patient TME status, prognosis and immunotherapy response. In this study, we identified distinct ICD-related modification patterns based on 158 TNBC cases in the TCGA-TNBC cohort. We then investigated the possible correlation between ICD-related modification patterns and TME cell infiltration characteristics in TNBC. By using univariate Cox and least absolute shrinkage and selection operator (LASSO) regression analysis, we created a risk scoring system (ICD score) to quantifiably evaluate the impact of ICD-related modification patterns in individual TNBC patient. Two different ICD-related modification patterns were found with significant differences in immune infiltration. Lower ICD score was correlated with higher immune infiltration, tumor mutational burden and significantly enriched in immune-related pathways, indicating a strong ability to activate immune response, which might account for relatively favorable prognosis of TNBC patients and could serve as a predictor to select suitable candidates for immunotherapy. We used two independent cohorts, GSE58812 cohort and Metabric cohort to validate prognosis and immunohistochemistry for preliminary in vitro validation. This study evidenced that the ICD-related modification patterns might exert pivotal roles in the immune infiltration landscape of TNBC and ICD score might act as potential predictors of prognostic assessment and immunotherapy response. This research provides unique insights for individualize immune treatment strategies and promising immunotherapy candidates screening.

Potential Value of HSP90α in Prognosis of Triple-Negative Breast Cancer.

BACKGROUND Triple-negative breast cancer (TNBC) is a distinct subtype of breast cancer, accounting for 12-18% of all breast cancer cases. It exhibits high heterogeneity and aggressiveness, resulting in a poorer prognosis with a high risk of early recurrence and metastasis. Due to the lack of expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2), as well as insensitivity to endocrine therapy, determining a standard treatment for TNBC is challenging. The identification of potential prognostic biomarkers is crucial for developing personalized treatment strategies for patients. MATERIAL AND METHODS Our study investigated the potential value of HSP90a in TNBC prognosis. A retrospective analysis was conducted on 127 TNBC patients and 127 Healthy controls from March 1, 2019 to July 31, 2022. Venous blood was collected and tested for HSP90alpha, CEA, CA199, and CA125, and we recorded the clinical characteristics of the patients, including age, BMI, alcohol consumption status, surgical history, CEA level, CA199 level, CA125 level, HSP90alpha level, tumor size, distant metastases, lymph node metastasis, and TNM stage. Univariate and multivariate methods were used to screen independent risk factors for progression-free survival (PFS) and overall survival (OS). RESULTS HSP90alpha is not only upregulated in TNBC but is also highly correlated with lymph node metastasis and TNM stage. The results of multivariate analysis showed that distant metastasis, TNM stage and HSP90a level were independent factors associated with PFS. BMI, tumor size, TNM stage, surgical history, and HSP90a level were independent factors influencing OS. CONCLUSIONS Our research findings demonstrate a significant association between high HSP90alpha expression and adverse clinical features, suggesting a poorer prognosis for TNBC patients.

Spindle apparatus coiled-coil protein 1 (SPDL1) serves as a novel prognostic biomarker in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has a poor prognosis, an ineffective diagnosis, and a high degree of aggressiveness. Therefore, novel therapeutic targets for TNBC urgently need to be identified. Through a series of bioinformatics analyses, including analysis of differential gene expression, protein-protein interaction (PPI) network, univariate cox regression, immune infiltration, pathway enrichment, etc, as well as auxiliary immunohistochemistry (IHC) and protein quantitativae analysis, to explore prognostic marker for TNBC. In TNBC tissues, we found that SPDL1 (CCDC99) was considerably overexpressed at both the mRNA and protein levels compared to that in normal and non-TNBC tissues. Additionally, we found that SPDL1-high expression was strongly linked to poor prognosis in TNBC patients. Excessive SPDL1 expression was positively correlated with tumor growth and strongly linked to the cell cycle, DNA replication, and the p53 signaling pathway. In addition, CIBERSORT analysis revealed that SPDL1 can affect the tumor immune microenvironment (TME) in TNBC, encourage the development of TNBC and act as a potential prognostic biomarker for TNBC. Patients with SPDL1-high expression were more sensitive to AZD8055. Notably, we discovered that SPDL1 is highly expressed in the majority of malignancies and may have an impact on the pancancer prognosis. SPDL1 can serve as a novel prognostic marker for TNBC and pancancer patients.

Fbxw7 suppresses carcinogenesis and stemness in triple-negative breast cancer through CHD4 degradation and Wnt/β-catenin pathway inhibition

BackgroundCancer stem cells (CSCs) are a small population of cells in tumor tissues that can drive tumor initiation and promote tumor progression. A small number of previous studies indirectly mentioned the role of F-box and WD repeat domain-containing 7 (FBXW7) as a tumor suppressor in Triple-negative breast cancer (TNBC). However, few studies have focused on the function of FBXW7 in cancer stemness in TNBC and the related mechanism.MethodsWe detected FBXW7 by immunohistochemistry (IHC) in 80 TNBC patients. FBXW7 knockdown and overexpression in MD-MBA-231 and HCC1937 cell models were constructed. The effect of FBXW7 on malignant phenotype and stemness was assessed by colony assays, flow cytometry, transwell assays, western blot, and sphere formation assays. Immunoprecipitation-Mass Spectrometry (IP-MS) and ubiquitination experiments were used to find and verify potential downstream substrate proteins of FBXW7. Animal experiments were constructed to examine the effect of FBXW7 on tumorigenic potential and cancer stemness of TNBC cells in vivo.ResultsThe results showed that FBXW7 was expressed at low levels in TNBC tissues and positively correlated with prognosis of TNBC patients. In vitro, FBXW7 significantly inhibited colony formation, cell cycle progression, cell migration, EMT process, cancer stemness and promotes apoptosis. Further experiments confirmed that chromodomain-helicase-DNA-binding protein 4 (CHD4) is a novel downstream target of FBXW7 and is downregulated by FBXW7 via proteasomal degradation. Moreover, CHD4 could promote the nuclear translocation of β-catenin and reverse the inhibitory effect of FBXW7 on β-catenin, and ultimately activate the Wnt/β-catenin pathway. Rescue experiments confirmed that the FBXW7-CHD4-Wnt/β-catenin axis was involved in regulating the maintenance of CSC in TNBC cells. In animal experiments, FBXW7 reduced CSC marker expression and suppressed TNBC cell tumorigenesis in vivo.ConclusionsTaken together, these results highlight that FBXW7 degrades CHD4 protein through ubiquitination, thereby blocking the activation of the Wnt/β-catenin pathway to inhibit the stemness of TNBC cells. Thus, targeting FBXW7 may be a promising strategy for therapeutic intervention against TNBC.

Long noncoding RNA LINC00885 upregulates NCK1 to promote cell viability and migration of triple-negative breast cancer cells through sponging miR-654-3p.

LINC00885 is a novel oncogenic long noncoding RNA (LncRNA) which is upregulated in various types of cancer, but its function in triple-negative breast cancer (TNBC) remains unknown. This study aimed to determine the role of LINC00885 on TNBC development. Clinical interrelation and survival analysis were determined using online database. The CCK-8 and Transwell assays were used to detect the proliferation and migration behaviors in TNBC cell lines. The interaction among genes was detected by RNA pull down assay. LncRNA LINC00885 was highly expressed in TNBC compared to normal breast like. Low levels of LINC00885 was related to good prognosis in TNBC patients compared to TNBC patients with high LINC00885. LINC00885-downregulation inhibited, whereas LINC00885-overexpression promoted the proliferation and migration capability of TNBC cell lines. In TNBC cell lines, noncatalytic region of tyrosine kinase 1 (NCK1) expression was positively associated with LINC00885 expression, and shRNA-mediated the depletion of NCK1 significantly abolished LINC00885 upregulation-mediated pro-tumor effects. Combined with online databases, miR-654-3p was screened as the direct target gene of LINC00885, which could directly bind to 3'-untranslated regions (3'-UTR) of NCK1, resulting in the decreased expression of NCK1 in TNBC cell lines. LINC00885 overexpression-mediated the upregulation of NCK1 was abrogated by miR-654-3p mimics. MiR-654-3p mimics significantly rescued the tumor promotive role caused by LINC00885-overexpression. However, exogenous NCK1 notably eliminated the anti-tumor effects caused by miR-654-3p mimics in LINC00885-overexpressed cells. LINC00885 is expressed at a high level in TNBC. LINC00885 promoted proliferation and migration by regulating the miR-654-3p/NCK1 axis in TNBC cell lines. Possibly, LINC00885 can be served as a potential therapeutic target for TNBC.

Epigenomic Profiling Advises Therapeutic Potential of Leukotriene Receptor Inhibitors for a Subset of Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype, with a poor survival rate compared to others subtypes. For a long time, chemotherapy was the only systemic treatment for TNBC, and the identification of actionable molecular targets might ultimately improve the prognosis for TNBC patients. We performed a genome-wide analysis of DNA methylation at CpG islands on a collection of one hundred ten breast carcinoma samples and six normal breast tissue samples using reduced representation bisulfite sequencing with the XmaI restriction enzyme (XmaI-RRBS) and identified a subset of TNBC samples with significant hypomethylation at the LTB4R/LTB4R2 genes' CpG islands, including CpG dinucleotides covered with cg12853742 and cg21886367 HumanMethylation 450K microarray probes. Abnormal DNA hypomethylation of this region in TNBC compared to normal samples was confirmed by bisulfite Sanger sequencing. Gene expression generally anticorrelates with promoter methylation, and thus, the promoter hypomethylation detected and confirmed in our study might be revealed as an indirect marker of high LTB4R/LTB4R2 expression using a simple methylation-sensitive PCR test. Analysis of RNA-seq expression and DNA methylation data from the TCGA dataset demonstrates that the expression of the LTB4R and LTB4R2 genes significantly negatively correlates with DNA methylation at both CpG sites cg12853742 (R = -0.4, p = 2.6 × 10-6; R = -0.21, p = 0.015) and cg21886367 (R = -0.45, p = 7.3 × 10-8; R = -0.24, p = 0.005), suggesting the upregulation of these genes in tumors with abnormal hypomethylation of their CpG island. Kaplan-Meier analysis using the TCGA-BRCA gene expression and clinical data revealed poorer overall survival for TNBC patients with an upregulated LTB4R. To this day, only the leukotriene inhibitor LY255283 has been tested on an MCF-7/DOX cell line, which is a luminal A breast cancer molecular subtype. Other studies compare the effects of Montelukast and Zafirlukast (inhibitors of the cysteinyl leukotriene receptor, which is different from LTB4R/LTB4R2) on the MDA-MB-231 (TNBC) cell line, with high methylation and low expression levels of LTB4R. In our study, we assess the therapeutic effects of various drugs (including leukotriene receptor inhibitors) with the DepMap gene effect and drug sensitivity data for TNBC cell lines with hypomethylated and upregulated LTB4R/LTB4R2 genes. LY255283, Minocycline, Silibinin, Piceatannol, Mitiglinide, 1-Azakenpaullone, Carbetocin, and Pim-1-inhibitor-2 can be considered as candidates for the additional treatment of TNBC patients with tumors demonstrating LTB4R/LTB4R2 hypomethylation/upregulation. Finally, our results suggest that the epigenetic status of leukotriene B4 receptors is a novel, potential, predictive, and prognostic biomarker for TNBC. These findings might improve individualized therapy for TNBC patients by introducing new therapeutic adjuncts as anticancer agents.

C9orf142 transcriptionally activates MTBP to drive progression and resistance to CDK4/6 inhibitor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) presents the most challenging subtype of all breast cancers because of its aggressive clinical phenotypes and absence of viable therapy targets. In order to identify effective molecular targets for treating patients with TNBC, we conducted an integration analysis of our recently published TNBC dataset of quantitative proteomics and RNA-Sequencing, and found the abnormal upregulation of chromosome 9 open reading frame 142 (C9orf142) in TNBC. However, the functional roles of C9orf142 in TNBC are unclear. In vitro and in vivo functional experiments were performed to assess potential roles of C9orf142 in TNBC. Immunoblotting, real-time quantitative polymerase chain reaction (RT-qPCR),and immunofluorescent staining were used to investigate the expression levels of C9orf142 and its downstream molecules. The molecular mechanisms underlying C9orf142-regulated mouse double minute 2 (MDM2)-binding protein (MTBP) were determined by chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays. In TNBC tissues and metastatic lymph nodes, we observed that C9orf142 exhibited abnormal up-regulation, and its elevated expression was indicative of unfavorable prognosis for TNBC patients. Both in vitro and in vivo functional experiments demonstrated that C9orf142 accelerated TNBC growth and metastasis. Further mechanism exploration revealed that C9orf142 transcriptionally activated MTBP, thereby regulating its downstream MDM2/p53/p21 signaling axis and the transition of cell cycle from G1 to S phase. Functional rescue experiment demonstrated that knockdown of MTBP attenuated C9orf142-mediated tumour growth and metastasis. Furthermore, depletion of C9orf142 remarkably increased the responsiveness of TNBC cells to CDK4/6 inhibitor abemaciclib. Together, these findings unveil a previously unrecognized effect of C9orf142 in TNBC progression and responsiveness to CDK4/6 inhibitor, and emphasize C9orf142 as a promising intervention target for TNBC treatment.

Nanoparticles (NPs)-mediated lncBCMA silencing to promote eEF1A1 ubiquitination and suppress breast cancer growth and metastasis.

Long non-coding RNAs (lncRNAs) play an important role in cancer metastasis. Exploring metastasis-associated lncRNAs and developing effective strategy for targeted regulation of lncRNA function invivo are of utmost importance for the treatment of metastatic cancer, which however remains a big challenge. Herein, we identified a new functional lncRNA (denoted lncBCMA), which could stabilize the expression of eukaryotic translation elongation factor 1A1 (eEF1A1) via antagonizing its ubiquitination to promote triple-negative breast cancer (TNBC) growth and metastasis. Based on this regulatory mechanism, an endosomal pH-responsive nanoparticle (NP) platform was engineered for systemic lncBCMA siRNA (siBCMA) delivery. This NPs-mediated siBCMA delivery could effectively silence lncBCMA expression and promote eEF1A1 ubiquitination, thereby leading to a significant inhibition of TNBC tumor growth and metastasis. These findings show that lncBCMA could be used as a potential biomarker to predict the prognosis of TNBC patients and NPs-mediated lncBCMA silencing could be an effective strategy for metastatic TNBC treatment.

Treatment Problems In Triple Negative Breast Cancer

Background: Breast cancer ranks second on the list of common diseases worldwide. It causes many deaths in the United States and Europe, second only to lung cancer. Triple-negative breast cancer (TNBC) does not express Estrogen Receptor (ER), Progesterone Receptor (PR), and Human Epithelial Receptor (HER2). It represents 24% of new cases of all breast cancer, and its incidence increases yearly. TNBC is a hormone-resistant breast cancer, so no current standard therapy exists. This article aims to explore regimen-resistant and troubleshooting treatment responses in TNBC cases. The method of writing this article is a literature review of studies using the keywords triple-negative breast cancer treatment and regimen, which are limited to only the most recent articles, 2012-2022, using search engines from PubMed, Science Direct, and Google Scholar. Results: There is no definitive therapy for the triple-negative breast cancer subtype, in which the TNBC type has no target receptor. Chemotherapy is the SOC of TNBC for early stage treatment. For late-stage and relapsed TNBC, however, chemotherapy is no longer the first choice. Currently there is no standard chemotherapy regimen that can be given to patients who experience relapse after chemotherapy because they will have a short response and lead to metastases. Some studies have shown that chemotherapy gives a better response, but the prognosis of TNBC remains poor. TNBC has different responses to therapy. TNBC showed a good response to combination chemotherapy along with pembrolizumab, capecitabine, olaparib, and radiotherapy, compared to chemotherapy by itself. Surgical therapy, such as Breast-Conserving Surgery (BCS), does not improve better prognosis in TNBC patients. Conclusions: Combining pembrolizumab, olaparib, capecitabine, and radiotherapy with chemotherapy increased survival rates compared to chemotherapy only.