TNBC Progression Research Articles

Small-molecule targeting BCAT1-mediated BCAA metabolism inhibits the activation of SHOC2-RAS-ERK to induce apoptosis of Triple-negative breast cancer cells

IntroductionTriple-negative breast cancer (TNBC) is the most malignant subtype of breast cancer with the worst prognosis. Exploring novel carcinogenic factors and therapeutic drugs for TNBC remains a focus to improve prognosis. Branched-chain amino acid transaminase 1 (BCAT1), a crucial enzyme in branched-chain amino acid (BCAA) metabolism, has been linked to various tumor developments, but its carcinogenic function and mechanism in TNBC remain unclear. Eupalinolide B (EB) is a naturally-derived small-molecule with anti-tumor activity, but its role in TNBC remains unknown. ObjectivesBy exploring the targets and pharmacological mechanisms of EB in inhibiting TNBC, this study aimed to discover novel therapeutic targets and potential inhibitors for TNBC, and elucidate novel pathogenic mechanisms of TNBC. MethodsThe inhibitory effect of EB on TNBC was investigated using mouse models and cellular phenotypic experiments. Activity-based protein profiling (ABPP) technology, pull down-WB, CETSA-WB and MST were utilized to discover and validate the targets of EB. The oncogenic role of BCAT1 was determined through clinical data analysis and biochemical experiments. To elucidate the mechanism by which EB inhibited TNBC, many methods, including but not limited to HPLC and proteomic sequencing were used. ResultsWe found that EB significantly inhibited TNBC progression. We identified BCAT1 as the direct target of EB and confirmed that BCAT1 was critical for TNBC development. EB inhibited BCAT1-involved BCAA metabolism to reduce the synthesis of BCAAs (including Leu, Ile, and Val), thereby inhibiting SHOC2 (a Leu-rich repeat protein) expression and the downstream SHOC2-participating RAS-ERK signaling pathway, ultimately leading to apoptosis of TNBC cells. ConclusionCollectively, this study not only elucidates the oncogenic role of BCAT1 and its downstream SHOC2-RAS-ERK signaling axis in TNBC progression but also opens up avenues for potential therapies targeting BCAT1 or BCAA metabolism (using EB alone or in combination with its inhibitor candesartan) for TNBC treatment.

Identification of the tumor metastasis-related tumor subgroups overexpressed NENF in triple-negative breast cancer by single-cell transcriptomics

Tumor metastasis is a continuous and dynamic process and is a major cause of tumor-related death in triple-negative breast cancer. However, this biological process remains largely unknown in triple-negative breast cancer. The emergence of single-cell sequencing enables a deeper understanding of the tumor microenvironment and provides a new strategy for discovering the potential mechanism of tumor metastasis. Herein, we integrated the single-cell expression profiling of primary and metastatic triple-negative breast cancer by Seurat package. Nine tumor cell subgroups were identified. Enrichment analysis suggested tumor subgroups (C0, C4) were associated with tumor metastasis with poor prognosis in TNBC. Weighted gene co-expression network was constructed and identified NENF was a metastasis-related gene. Subsequently, RT-qPCR, Immunohistochemistry, and western blot confirmed NENF is highly expressed in TNBC tissues. And cell function assays indicated NENF promote cell invasion and migration through regulating EMT in TNBC. Finally, TIDE and Connectivity Map database suggest the candidate drugs for targeting NENF. In conclusion, our findings provide a new insight into the progression and metastasis of TNBC and uncover NENF may be a prognostic biomarker and potential therapy targets.

LINC01503 promotes the cell proliferation, migration and invasion of triple-negative breast cancer as a ceRNA to elevate SPNS2 expression by sponging miR-335–5p

ObjectiveTriple-negative breast cancer (TNBC) is a common cancer with high aggressiveness and high mortality in women. Recently, a plenty of studies have indicated that long non-coding RNAs (lncRNAs) exert the crucial function in human cancers, TNBC is included. The carcinogenicity of lncRNA long intergenic non-protein coding RNA 1503 (LINC01503) has been confirmed in several cancers, nevertheless, its function in TNBC still unclear. Therefore, our study aimed to reveal the underlying mechanism of LINC01503 in TNBC. MethodsIn our study, RT-qPCR was performed to detect the expression of LINC01503 in TNBC cells. The proliferative, invasive, migratory and apoptotic abilities of TNBC cells were detected by functional assay such as CCK-8, clone formation, EdU staining, transwell, and flow cytometry. RIP, RNA pull down, and luciferase assay revealed interactions between LINC01503, miR-335–5p, and sphingolipid transporter protein 2 (SPNS2). Finally, rescue experiments were performed to validate the previous results. ResultsLINC01503 expression was singularly high in TNBC cells. LINC01503 knockdown could restrain cell proliferation, invasion and migration, but accelerated cell apoptosis in TNBC. What’s more, miR-335–5p could be sponged by LINC01503 in TNBC. We also found that overexpressed miR-335–5p could inhibit cell proliferation, migration and invasion and facilitates cell apoptosis. Moreover, SPNS2 was the target gene of miR-335–5p and it functioned as an oncogene in TNBC cells. Finally, we found that overexpressed SPNS2 or inhibited miR-335–5p could reverse the suppressive function of silencing LINC01503 on TNBC progression. ConclusionLINC01503 could facilitate cell proliferation, migration and invasion of TNBC by sponging miR-335–5p to elevate SPNS2 expression.

SPI1-mediated transcriptional activation of CEP55 promotes the malignant growth of triple-negative breast cancer and M2 macrophage polarization

BackgroundTriple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks the expression of three receptors commonly targeted in breast cancer treatment. In this study, the research focused on investigating the role of centrosomal protein 55 (CEP55) in TNBC progression and its interaction with the transcription factor Spi-1 proto-oncogene (SPI1). MethodsVarious techniques including qRT-PCR, western blotting, and immunohistochemistry assays were utilized to examine gene expression patterns. Functional assays such as wound-healing assay, transwell invasion assay, 5-Ethynyl-2’-deoxyuridine assay, and metabolic assays were conducted to assess the impact of CEP55 on the behaviors of TNBC cells. CD163-positive macrophages were quantified by flow cytometry. The chromatin immunoprecipitation assay and dual-luciferase reporter assay were performed to assess the association of SPI1 with CEP55. A xenograft mouse model experiment was used to analyze the impact of SPI1 on tumor development in vivo. ResultsCEP55 and SPI1 expression levels were significantly upregulated in TNBC tissues and cells. The depletion of CEP55 led to decreased TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization, indicating its crucial role in promoting TNBC progression. Moreover, SPI1 transcriptionally activated CEP55 in TNBC cells, and its overexpression was associated with accelerated tumor growth in vivo. Further, CEP55 overexpression relieved SPI1 silencing-induced inhibitory effects on TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization. ConclusionSPI1-mediated transcriptional activation of CEP55 plays a key role in enhancing TNBC cell migration, invasion, proliferation, glucose metabolism, and M2 macrophage polarization. These insights provide valuable information for potential targeted therapies to combat TNBC progression by modulating the SPI1-CEP55 axis.

GluOC promotes proliferation and metastasis of TNBC through the ROCK1 signaling pathway

BackgroundTriple negative breast cancer (TNBC) is a type of breast cancer that is negative for oestrogen receptor, progesterone receptor and human epidermal growth factor receptor 2, is highly malignant and aggressive, lacks of corresponding targeted therapy, and has a relatively poor prognosis. Therefore, understanding the mechanism of TNBC development and formulating effective treatment strategies for inducing cell death are still urgent tasks in the treatment of TNBC. Research has shown that uncarboxylated osteocalcin can promote the proliferation of prostate cancer, lung adenocarcinoma and TNBC cells, but the mechanism by which GluOC affects TNBC growth and metastasis needs further study.MethodsMDA-MB-231 breast cancer cells were used for in vitro cell analysis. Key target molecules or pathways were identified by RNA sequencing, and migration ability was detected by scratch assays, Transwell assays, cell adhesion assays and western blot analysis. Fluorescence staining, colony detection, qRT‒PCR and flow cytometry were used to detect apoptosis, oxidative stress, the cell cycle and the stemness of cancer cells, and a xenotransplantation model in BALB/C nude mice was used for in vivo analysis.ResultsThis study demonstrated that GluOC facilitates the migration of MDA-MB-231 breast cancer cells through the ROCK1/MYPT1/MLC2 signalling pathway and promotes the proliferation of TNBC cells via the ROCK1/JAK2/PIK3CA/AKT signalling pathway. Experiments in nude mice demonstrated that GluOC promoted tumour cell proliferation and metastasis in tumour-bearing mice, which further clarified the molecular mechanism of TNBC growth and invasion.ConclusionOur findings highlight the importance of GluOC in driving TNBC progression and its association with poor patient outcomes. This study clarifies the functional effects of GluOC on TNBC growth, providing insight into the molecular basis of TNBC and potentially providing new ideas for developing targeted therapies to improve patient outcomes.

Advancing targeted combination chemotherapy in triple negative breast cancer: nucleolin aptamer-mediated controlled drug release

BackgroundTriple-negative breast cancer (TNBC) is a recurrent, heterogeneous, and invasive form of breast cancer. The treatment of TNBC patients with paclitaxel and fluorouracil in a sequential manner has shown promising outcomes. However, it is challenging to deliver these chemotherapeutic agents sequentially to TNBC tumors. We aim to explore a precision therapy strategy for TNBC through the sequential delivery of paclitaxel and fluorouracil.MethodsWe developed a dual chemo-loaded aptamer with redox-sensitive caged paclitaxel for rapid release and non-cleavable caged fluorouracil for slow release. The binding affinity to the target protein was validated using Enzyme-linked oligonucleotide assays and Surface plasmon resonance assays. The targeting and internalization abilities into tumors were confirmed using Flow cytometry assays and Confocal microscopy assays. The inhibitory effects on TNBC progression were evaluated by pharmacological studies in vitro and in vivo.ResultsVarious redox-responsive aptamer-paclitaxel conjugates were synthesized. Among them, AS1411-paclitaxel conjugate with a thioether linker (ASP) exhibited high anti-proliferation ability against TNBC cells, and its targeting ability was further improved through fluorouracil modification. The fluorouracil modified AS1411-paclitaxel conjugate with a thioether linker (FASP) exhibited effective targeting of TNBC cells and significantly improved the inhibitory effects on TNBC progression in vitro and in vivo.ConclusionsThis study successfully developed fluorouracil-modified AS1411-paclitaxel conjugates with a thioether linker for targeted combination chemotherapy in TNBC. These conjugates demonstrated efficient recognition of TNBC cells, enabling targeted delivery and controlled release of paclitaxel and fluorouracil. This approach resulted in synergistic antitumor effects and reduced toxicity in vivo. However, challenges related to stability, immunogenicity, and scalability need to be further investigated for future translational applications.

Quercetin inhibits chronic stress-mediated progression of triple-negative breast cancer by blocking β2-AR/ERK1/2 pathway

Chronic stress-mediated sustained release of neurotransmitters, which ultimately leads to the activation of β2-adrenergic receptor (β2-AR) signaling, is one of the most important reasons for triple-negative breast cancer (TBNC) progression. Quercetin (Que) has been proven to have the advantage of ameliorating stress psychological disorder. Our present study aimed to investigate the effect of Que on tumor growth and metastasis in TNBC xenograft mice undergoing stress, and to explore its underlying mechanisms. We first evaluated the effect of Que on the progression of TNBC in nude mice in vivo. The results showed that, Que could inhibit chronic stress-induced TNBC growth and occurrence of lung metastasis. We subsequently employed epinephrine (E) as a representative of stress hormone to investigate its possible mechanism in vitro. The results showed that, Que could inhibit E-mediated proliferation and migration of TNBC cells by blocking β2-AR/ERK1/2 pathway. In conclusion, our data demonstrated that Que could inhibit chronic stress-induced ERK1/2 activity in TNBC cells, and thereby weakening the potential for TNBC growth and metastasis.

Constructing a novel prognostic model for triple-negative breast cancer based on genes associated with vasculogenic mimicry.

Research has shown a connection between vasculogenic mimicry (VM) and cancer progression. However, the functions of genes related to VM in the emergence and progression of TNBC have not been completely elucidated. A survival risk model was constructed by screening biomarkers using DESeq2 and WGCNA based on public TNBC transcriptome data. Furthermore, gene set enrichment analysis was performed, and tumor microenvironment and drug sensitivity were analyzed. The selected biomarkers were validated via quantitative PCR detection, immunohistochemical staining, and protein detection in breast cancer cell lines. Biomarkers related to the proliferation and migration of TNBC cells were validated via in vitro experiments. The findings revealed that 235 target genes were connected to the complement and coagulation cascade pathways. The risk score was constructed using KCND2, NRP1, and VSTM4. The prognosis model using the risk score and pathological T stage yielded good validation results. The clinical risk of TNBC was associated with the angiogenesis signaling pathway, and the low-risk group exhibited better sensitivity to immunotherapy. Quantitative PCR and immunohistochemistry indicated that the expression levels of KCND2 in TNBC tissues were higher than those in adjacent nontumor tissues. In the TNBC cell line, the protein expression of KCND2 was increased. Knockdown of KCND2 and VSTM4 inhibited the proliferation and migration of TNBC cells in vitro. In this study, three VM-related biomarkers were identified, including KCND2, NRP1, and VSTM4. These findings are likely to aid in deepening our understanding of the regulatory mechanism of VM in TNBC.

Chemotherapy-elicited extracellular vesicle CXCL1 from dying cells promotes triple-negative breast cancer metastasis by activating TAM/PD-L1 signaling

BackgroundTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, and chemotherapy still serves as the cornerstone treatment functioning by inducing cytotoxic cell death. Notably, emerging evidence suggests that dying cell-released signals may induce cancer progression and metastasis by modulating the surrounding microenvironment. However, the underlying molecular mechanisms and targeting strategies are yet to be explored.MethodsApoptotic TNBC cells induced by paclitaxel or adriamycin treatment were sorted and their released extracellular vesicles (EV-dead) were isolated from the cell supernatants. Chemokine array analysis was conducted to identify the crucial molecules in EV-dead. Zebrafish and mouse xenograft models were used to investigate the effect of EV-dead on TNBC progression in vivo.ResultsIt was demonstrated that EV-dead were phagocytized by macrophages and induced TNBC metastasis by promoting the infiltration of immunosuppressive PD-L1+ TAMs. Chemokine array identified CXCL1 as a crucial component in EV-dead to activate TAM/PD-L1 signaling. CXCL1 knockdown in EV-dead or macrophage depletion significantly inhibited EV-dead-induced TNBC growth and metastasis. Mechanistic investigations revealed that CXCL1EV-dead enhanced TAM/PD-L1 signaling by transcriptionally activating EED-mediated PD-L1 promoter activity. More importantly, TPCA-1 (2-[(aminocarbonyl) amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide) was screened as a promising inhibitor targeting CXCL1 signals in EVs to enhance paclitaxel chemosensitivity and limit TNBC metastasis without noticeable toxicities.ConclusionsOur results highlight CXCL1EV-dead as a novel dying cell-released signal and provide TPCA-1 as a targeting candidate to improve TNBC prognosis.Graphical abstract

Exosomal miR-182-5p from breast cancer cells reprogram tumor-associated macrophages and promotes TNBC progression by targeting Notch1 in macrophages

IntroductionThe regulatory role of miR-182 in breast cancer malignancy and macrophage reprogramming is well-established. However, the mechanisms through which miR-182 overexpression in tumor cells influences macrophage polarization remain elusive.Material and methodsAfter transfection with miR-182-5p mimics, inhibitors, and controls for 24 hours, exosomes were extracted by differential centrifugation from transfected MDA-MB-231. Macrophages co-cultured with these exosomes to illustrate the regulative effects of exo-miR-182-5p reprogram macrophage. Furthermore, breast cancer cells co-cultured with exo-miR-182-5p reprogrammed M2 macrophages to demonstrate the effects of reprogrammed M2 macrophages to influence breast cancer progression. After all, these findings were validated in cell derived xenograft (CDX) BALB/C nude-mice model.ResultsIn this study, we demonstrate that exosome-derived miR-182-5p from TNBC cells reprograms M2 macrophage polarization through direct combined with Notch1, thereby enhancing breast cancer progression in vitro and in vivo. When co-cultured with exosomes from TNBC cells transfected with miR-182-5p mimics or inhibitors, macrophages showed altered Notch1/Hes1 pathway expression, leading to M2 polarization and subsequent changes in reactive oxygen species (ROS), inflammation, and other biochemical markers. Furthermore, breast cancer cells co-cultured with exosomes reprogrammed macrophages exhibited increased colony formation, migration, and invasion, as well as reduced apoptosis. These findings were validated in a BALB/C nude-mice model.ConclusionsThis study pioneers the elucidation of the feedback loop mechanism between breast cancer cells and macrophages mediated by the exosome-derived miR-182-5p/Notch1 pathway, highlighting its role in macrophage reprogramming. Although the therapeutic application of miR-182-5p inhibitors as anticancer agents remains in the early stages, targeting macrophage polarization represents a promising avenue for breast cancer therapy.

Novel combinatorial autophagy inhibition therapy for triple negative breast cancers

BackgroundTriple negative breast cancer (TNBC) has the worst prognosis among breast cancer subtypes. It is characterized by lack of estrogen, progesterone and human epidermal growth factor 2 receptors, and thus, have limited therapeutic options. Autophagy has been found to be correlated with poor prognosis and aggressive behaviour in TNBC. This study aimed to target autophagy in TNBC via a novel approach to inhibit TNBC progression. MethodsImmunoblotting and confocal microscopy were carried out to examine the effect of tumor microenvironmental stressors on autophagy. Cellular proliferation and migration assays were used to test the effect of different autophagy inhibitors and standard chemotherapy alone or in combination. In vivo xenograft mouse model was utilized to assess the effect of autophagy inhibitors alone or in combination with Paclitaxel. High resolution mass spectrometry based proteomic analysis was performed to explore the mechanisms behind chemoresistance in TNBC. Lastly, immunohistochemistry was done to assess the correlation between autophagy related proteins and clinical characteristics in TNBC tissue specimens. ResultsMetabolic stressors were found to induce autophagy in TNBC cell lines. Autophagy initiation inhibitors, SAR405 and MRT68921, showed marked synergy in their anti-proliferative activity in both chemosensitive and chemoresistant TNBC cell models. Paradoxically, positive expression of autophagosome marker LC3 was shown to be associated with better overall survival of TNBC patients. ConclusionIn this study, a novel combination between different autophagy inhibitors was identified which inhibited tumor cell proliferation in both chemosensitive and chemoresistant TNBC cells and could result in development of a novel treatment modality against TNBC.

Abstract LB188: PEA-15 phosphorylation mediates pro-tumor effects by promoting β-catenin activity in TNBC

Abstract Triple negative breast cancer (TNBC) is a subtype of breast cancer that is defined by a lack of estrogen receptor, progesterone receptor, and HER2-neu gene product expression. Previously, we found that TNBC cells with phosphorylated PEA-15 adaptor protein exhibited decreased clonogenic and anchorage-independent growth in vitro, and decreased growth and lung metastasis in vivo. Further, we found lower levels of phosphorylated β-catenin at S675 in PEA-15-AA (non-phosphorylatable) mutants than in PEA-15-DD (phosphomimetic) mutants. We therefore hypothesized that phosphorylated PEA-15 enhances the aggressiveness of TNBC by regulating the phosphorylation of β-catenin at S675. We first screened a panel of TNBC cells for β-catenin levels via western blot. Next to assess the biological role of β-catenin we knocked down its expression in TNBC cells (SUM149, BT549). In the β-catenin knockouts, proliferation decreased by 20%(P<0.0001) mammosphere formation by 69% (P<0.001), and clonogenic growth by 65% (P<0.0001), when compared to the vector control. To determine if PEA-15 mediates pro-tumor effects by phosphorylating β-catenin at S675, we created a nonphosphorylatable (S675A) and a phosphomimetic (S675D) form of β-catenin. We transfected BT549 cells with S675A and observed a decrease of 26% (P=0.011) in proliferation, 65% (P<0.001) in mammospheres, and 54% (P=0.0001) in clonogenic formation when compared with WT β-catenin transfected cells. Next, we transfected SUM149 cells with S675D and found an increase of 58% (P<0.001) in proliferation, 79% (P=0.001) in mammospheres, and 23% (P=0.0303) in clonogenic formation when compared with WT β-catenin transfected cells. To test if PEA-15 phosphorylation mediates β-catenin activity, we transfected the S675A and S675D form of β-catenin into MDA-MB-468 cell lines which stably express PEA-15-AA and PEA-15-DD. When we performed a rescue experiment with PEA-15-AA cells transfected with S675D we found a 78% (P=0.0008) increase in mammospheres when compared to vector transfected cells. Conversely, when PEA-15-DD cells were transfected with S675A we found a 53% (P=0.0004) decrease in mammospheres when compared to vector transfected cells. Next, we did a multiplex staining of a tissuemicroarray (n= 102 human TNBC tumors). We observed that in patients with grade 3 tumors, the percentages of cells expressing total PEA-15(P=0.005), P-PEA-15-S116(P=0.038), and p-β-catenin (P=0.0005) were significantly higher than those in patients with grade 2 tumors. Together, these results show that PEA-15 phosphorylation mediates tumor aggressiveness in TNBC by promoting β-catenin activity. Future studies will evaluate the clinical significance of PEA-15 and β-catenin and their phosphorylated forms in TNBC patients. These results will establish β-catenin and PEA-15 phosphorylation as potential markers of, and targets against TNBC progression. Citation Format: Alex W. Tan, Mohd Mughees, Wendy A. Woodward, Debu Tripathy, Natalie Fowlkes, Chandra Bartholomeusz. PEA-15 phosphorylation mediates pro-tumor effects by promoting β-catenin activity in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB188.

Abstract 5681: Identification of dysregulated miRNAs involved in TNBC progression

Abstract Triple negative breast cancer (TNBC) is the most aggressive type of all breast cancer subtypes. Although various studies have been performed to identify prognostic and therapeutic targets for TNBC, the TNBC specific biomarkers have not yet been elucidated. Herein, we identified dysregulated miRNAs in TNBC via analysis of miRNA microarray in breast cancer tissues and found the oncogenic RNA (miR-374a-5p) and tumor suppressive miRNAs (miR-320c, miR-371b-5p). These miRNAs were closely related to overall survival of TNBC patients and status and involved in TNBC progression or drug responsiveness. Collectively, these results suggest that dysregulated miRNAs might serve as potential prognostic marker or therapeutic target for TNBC. Citation Format: Minyoung Lee, Yesol Kim, Dasom Son, Sera Lim, Jong Hoon Park. Identification of dysregulated miRNAs involved in TNBC progression [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 5681.

Abstract 6969: Determining the role of obesity in breast cancer progression

Abstract Breast cancer, especially triple negative breast cancer, impacts a large proportion of the female population. Approximately ten to fifteen percent of all breast cancer diagnoses will be triple negative. Triple negative breast cancer lacks receptors that are used as therapeutic targets for other forms of breast cancer and has the poorest prognosis. Notably, the age of onset has also decreased, which correlates with the rise in prevalence of obesity. In addition, past studies revealed that maintaining a high BMI from childhood correlates with higher cancer mortality in adulthood. However, the mechanisms by which obesity contributes to cancer progression are not clear. Adipose tissue from different locations (visceral vs. subcutaneous, abdominal vs. femoral) and from patients with obesity from childhood rather than as adults could have different molecular characteristics. Further, depending on the genetic background, different profiles may be more susceptible to environmental cues that could drive their progression. To address this, we are using conditioned media collected from tissues that reflect these differences and determining how they affect TNBC cell lines with distinct genetic profiles. Preliminary data revealed that adipose tissue from both the abdominal and femoral regions of high BMI individuals caused an increase in the proliferation rate of TNBC cell lines. To determine if the adipocytes vs. other cell types in the adipose tissue drive this growth, we are co-culturing mature adipocytes from these tissues with the TNBC cell lines. Recent studies by other groups showed that adipocyte-derived extracellular vesicles could drive the expression of EMT genes in cancer cells. We will purify EVs from the adipocytes and characterize differences in their molecular signatures for different patients to determine which factors cause the change in TNBC progression and correlate this with different fat depots and/or age of onset. We will also measure the effect of adipose tissue and/or adipocytes on other cancer hallmarks including metastasis (migration and invasion) and changes in metabolism. These studies should reveal how adipocytes confer changes in TNBC progression, and how to consider obesity in different treatment regimes. Citation Format: Alexandra Perlman, Alisa Piekny, Elena Kuzmin, Sylvia Santosa. Determining the role of obesity in breast cancer progression [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 6969.

Abstract 2139: Identifying molecular mechanisms in triple negative breast cancer disparities: Unveiling the role of Hedgehog signaling in non-Hispanic Black women

Abstract Triple negative breast cancer (TNBC) represents an aggressive and therapeutically challenging breast cancer subtype with higher incidence and mortality rates in non-Hispanic Black (NHB) women. This disparity persists even after adjusting for socioeconomic factors, tumor biology, and quality of care. The study aims to unravel specific molecular mechanisms underlying this disparity, with a focus on Hedgehog signaling pathways in premenopausal NHB women. Understanding these mechanisms is vital for developing targeted therapies and personalized medicine approaches to address these health disparities. Our research utilized a unique cohort of NHB and non-Hispanic White (NHW) women who underwent breast reduction surgery at the Mayo Clinic and donated breast tissue samples for our study. We isolated RNA from whole tissue sections from a cohort of 12 NHB and 40 NHW women who were parous and premenopausal at time of surgery. We performed gene expression profiling using NanoString BC360 and IO360 assays. This allowed us to identify a distinct gene expression signature characteristic of basal-like/TNBC and indicative of heightened activity in the Hedgehog signaling pathway. We also employed advanced cell culture techniques to generate tissue organoids and subculture human mammary epithelial cells (HMECs) that preserve the intrinsic characteristics of the original tissue samples. We found that significantly differential expression of IL20RA and SOX10, principal factors in the gene expression signature, were preserved in both the derived organoids and the HMECs. These results suggest a potential intrinsic difference in the activation of the Hedgehog pathway in the breast epithelial cells of NHB women. This activation may contribute to increased proliferation and transformation of basal stem cells within the breast, possibly fueling the development and progression of TNBC. The implication of activated Hedgehog signaling in NHB women opens new avenues for understanding TNBC disparities. IL20RA and SOX10 represent potential biomarkers for TNBC risk that may serve as a stratification tool for NHB women. The results of this study have the potential to improve assessment and management of TNBC risk among NHB women, thereby contributing significantly to reducing health disparities in breast cancer outcomes. Citation Format: Jennifer Vanessa Cabezas, Savanna Touré, Matthew Wilson, Melody L. Stallings Mann, Laura M. Pacheco-Spann, Joshua W. Ogony, Mark E. Sherman, Derek C. Radisky. Identifying molecular mechanisms in triple negative breast cancer disparities: Unveiling the role of Hedgehog signaling in non-Hispanic Black women [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 2139.

Abstract 650: Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast cancer

Abstract Triple-negative Breast cancer (TNBC) is characterized by the absence of ER, PR, and HER-2 receptors and is associated with poor prognosis, early relapse, and distant metastasis. In neoadjuvant setting, most patients (~50-70%) with TNBC do not achieve complete remission. Currently, there is no effective targeted therapy for TNBC patients. Therefore, identifying novel molecular targets and developing alternative therapeutic strategies are urgently needed. PIM3 is a serine/threonine kinase and is upregulated in leukemia and various solid cancers. Although PIM1 has been shown to play a critical role in cell proliferation in cMyc-enriched TNBC tumors, the role of PIM3 in TNBC is not known. To determine the clinical significance of PIM3 in TNBC patient prognosis and progression, we performed a Kaplan-Meier analysis in the TCGA TNBC patient database. We found that PIM3 expression is significantly associated with shorter patient survival in TNBC patients (n=77, p=0.0088). PIM3 protein is highly expressed in TNBC cells compared to normal breast epithelium and other ER+ and HER2+ breast cancer subtypes. Inhibition of PIM3 by siRNA significantly suppressed TNBC cell proliferation, migration, and invasion of TNBC cells including MDA-MB-231, MDA-MB-436, and BT20 cells. Lenti-based PIM3 overexpression induced TNBC cell proliferation, invasion, and migration. Non-biased reverse phase protein array (RPPA) assay and Western blot analysis showed that PIM3 inhibition markedly reduced the expression of EF2 Kinase protein, and we found it to be associated with shorter patient survival and poor prognosis. Immunoprecipitation of PIM3 indicated that it forms a heterodimer with EF2K whose inhibition by siRNA also suppressed TNBC cell proliferation, migration, invasion, and tumor growth. Furthermore, PIM3 inhibition by siRNA significantly enhanced the antiproliferative effect of Doxorubicin, which is one of the first-line chemotherapeutics used in patients. Moreover, in vivo therapeutic delivery of PIM3 siRNA loaded albumin nanoparticles (ALNPs-PIM3 siRNA) markedly delayed tumor growth of two different TNBC tumor xenograft models (MDA-MB-231 and MDA-MB-436) in mice. Analysis of tumors by IHC after 4 weeks of treatment demonstrated that ALNP-PIM3 siRNA treatment led to inhibition of intra-tumoral proliferation and induction of apoptosis. Currently, we are conducting an in vivo study in TNBC tumor models by combining ALNP-PIM3 siRNA therapy with doxorubicin. Overall, our studies suggest that PIM3 is a marker for poor patient survival that drives TNBC tumor growth and progression and a novel potential molecular target in TNBC. Citation Format: Pinar Atalay, Goknur Kara, Rumeysa Ozyurt, Nermin Kahraman, Bulent Ozpolat. Pim3 kinase is a poor prognostic marker and novel molecular target for triple negative breast cancer [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 650.

Microarray analysis of differentially expressed miRNA in triple negative breast cancer: A study of western India

BackgroundTriple-negative breast cancer (TNBC) is a genetically and morphologically heterogeneous group with aggressive biological behaviour and specific response to therapy. It accounts for 15–20% of all breast cancers and has two subtypes: basal like and non-basal like. MicroRNAs, which regulate gene expression, play a role in TNBC, potentially acting as oncogenes or tumor suppressors. ObjectiveIdentification of differentially expressed miRNA of potential clinical relevance in TNBC patients. MethodsIn this study, miRNA profiling by microarray was performed in tumor tissues of 86 patients with TNBC and 12 healthy individual. Further, the clinical relevance of differentially expressed miRNA was evaluated. ResultsIn TNBC, 2410 differentially expressed miRNAs were identified and of them 98% were down-regulated, while only 2% were up-regulated. Up regulation of 55 miRNA was observed which target 16 genes. Top 5 genes identified were CDNK1A, p53, TGFB1, APC and HRAS. Of 7 ranking methods, 5 ranking method identified TGFB1 as most significant hub gene. Up regulated miRNA expression then compared between patients who undergone remission and patients who developed disease relapse and only miR-4532 was found upregulated in patients with disease relapse. Further, up regulation of miR-4532 showed a trend of reduced disease-free and overall survival. The down-regulated miRNAs target 238 genes involved in TNBC pathogenesis and progression. The top five hub genes were CDH1, PTEN, MYC, STAT3, and VEGFA. Of 7 ranking methods, 5 ranking method identified STAT3 as most significant hub gene. This study identified 32 novel miRNAs playing a tumor suppressive role and found down-regulated in TNBC. Among these two novel miRNAs, miR-1273g-3p and miR-4459 were found expressed in all TNBC patients. Patients with down-regulation of these miRNAs showed significantly reduced disease-free and overall survival. The ROC curve analysis indicated that miR-4532 and miR-4459 were successful in distinguishing TNBC patients from healthy controls. ConclusionOur data identified that up regulation of miR-4532 and down regulation of miR-4459 might have the potential to be used as both diagnostic and prognostic biomarker in TNBC.

Abstract 2939: Harnessing in vivo CRISPR screen to uncover metabolic dependencies during cancer progression

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer, with the worst prognosis and limited treatment options. TNBC cells undergo metabolic reprogramming in response to nutrient limitations and changes in the tumor microenvironment throughout cancer progression. In the early stages, tumor cells are found to prioritize nutrient uptake and biosynthesis, but as they grow, intensified competition for nutrients fosters the emergence of subpopulations of cancer cells that excel in utilizing specific nutrients. The substantial metabolic pathways and genes that regulate each phase of cancer progression remain poorly understood. To address this, we have developed a stable integration, site-specific, and inducible CRISPR-Cas9 system that allows for temporal control of genetic perturbation. We conducted an unbiased in vivo loss-of-function CRISPR screen aimed at identifying metabolic vulnerabilities at different phases of cancer progression of TNBC. The inducible CRISPR system was activated only at the desired study period of each phase, thus capable of uncovering metabolic dependencies for TNBC proliferation at each distinct phase of cancer progression. As many large-scale CRISPR screens have previously been performed to identify gene dependencies for cancer progression in vitro, our CRISPR screen could address the shortcomings of in vitro screens by identifying novel essential metabolic genes that are significantly important for cancer growth in vivo. Preliminary data from our CRISPR screen revealed different pathways to be significantly important for tumor growth and proliferation at different phases of TNBC progression. For example, we find that ferroptosis is altered during the early lag phase of TNBC cancer progression, whereas, at the later stage of cancer progression, metastatic tumors upregulate serine metabolism, suggesting that secondary tumors could have distinct metabolism from the primary sites. Altogether, the findings from this study could provide valuable insights into stepwise metabolic dependencies arising from microenvironmental limitations and identify potential therapeutic targets at each phase of cancer progression. Moreover, our CRISPR-Cas9 model holds promise for dissecting the complex mechanisms of other cancer progressions. Citation Format: Seow Qi Ng, Zhengwei Wu, Jessica A. Lidster, Wai Leong Tam. Harnessing in vivo CRISPR screen to uncover metabolic dependencies during cancer progression [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 2939.

Abstract 3067: SLC7A5/LAT1 promotes the Warburg Effect for TNBC progression through Trp/QPRT/NAD+ pathway

Abstract SLC7A5/LAT1 is a cross-membrane transporter that functions in uptake of large neutral amino acids into cells. Many pathological studies have shown that LAT1 is highly expressed in cancer tissue from various origins such as lung, biliary tract, breast, prostate, bone, and their metastatic legions. High LAT1 expression has been recognized as a significant prognostic marker in various cancers. Our TCGA data analysis showed that the expression level of LAT1 is higher in TNBC tumors compared with normal breast tissues. The higher expression level of LAT1 is linked with poor overall and progression-free survival. Dysregulation of amino acid transporters lead to metabolic reprogramming, which changes intracellular amino acid levels, contributing to the pathogenesis of cancer. However, it remains elusive whether and how LAT1 re-wires the cellular metabolic programs to promote TNBC tumor proliferation. In this study, we showed that inhibition of SLC7A5/LAT1 significantly reduced TNBC cell proliferation, viability, migration capability while promoting apoptosis. Mechanistically, we found significant positive correlations of LAT1 with PKM2 and LDHA in breast cancer tissues, which are key enzymes in mediating glycolysis and lactate fermentation or the Warburg Effect. Knock down LAT1 via siRNA decreased the expression levels of p-PKM2 and p-LDHA in TNBC cells. Tryptophan, a major substrate amino acid transported into cytosol by LAT1, is the precursor for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). The enzyme quinolinic acid phosphoribosyltransferase (QPRT) catalyzes the conversion from quinolinate, a product of tryptophan degradation, to NAD+. We showed LAT1 inhibition reduced the expression level of QPRT, cytosolic NAD+/NADH ratio as well as lactate production. Addition of L-Tryptophan increased the levels of p-PKM2 and p-LDHA while knockdown of LAT1 reversed tryptophan-mediated increase of PKM2 and LDHA activities, suggesting that LAT1 may regulate PKM2/LDHA through Trp/QPRT/NAD+ pathway for promoting the Warburg Effect. Additionally, we found upregulations of SLC7A5/LAT1, p-PKM2 and p-LDHA in doxorubicin-resistant cells and PDX mouse model compared with their corresponding controls. Collectively, these results provide evidence that overexpression of LAT1 in TNBC drives cancer progression and drug resistance by reinforcing the Warburg Effect, suggesting LAT1 as a promising therapeutic target for addressing TNBC. Our ongoing work will evaluate whether inhibiting LAT1 genetically or pharmacologically reduces xenograft tumor growth and enhances the sensitivities of TNBC resistant cells to chemotherapy in vivo. Our investigation will elucidate the underlying mechanisms involved in these processes. Citation Format: Margot Y. Fedoroff, Lei Zhao, Leili Saeednejad Zanjani, Jun He. SLC7A5/LAT1 promotes the Warburg Effect for TNBC progression through Trp/QPRT/NAD+ pathway [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 3067.

Abstract 5728: YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors

Abstract Metastatic breast cancer (BC) is the 2nd leading cause of death in women in the US, annually accounting for more than 43,000 deaths and 281,000 new cases of invasive BC. Amongst genetically distinct BC subtypes, those classified as being “triple-negative” (TNBC) are especially devastating due to their highly metastatic behavior, their propensity to recur rapidly, and their low response to standard-of-care therapies. In fact, the acquisition of chemoresistant phenotypes represents the main cause of disease recurrence, metastasis, and death in TNBC patients. Currently, the molecular mechanisms that regulate TNBC progression and metastasis remain unknown, as does the way these metastatic tumors acquire resistance to standard-of-care therapies. We recently established YB1 as a novel driver of these deadly TNBC activities, doing so by stimulating the cancer stem cell phenotype, and by disrupting normal cell cycle progression, therefore promoting therapy resistance and metastasis of TNBC tumors. YB1 is a multifunctional protein that acts as a transcription factor of cancer stem cell genes Nanog, Oct and Sox. Moreover, aberrant activation of YB1 contributes to the metastatic progression of several cancers, including TNBC. Our investigations revealed a major role of YB1 in the regulation of several hallmarks of cancer that drive TNBC tumors progression and metastasis, both in vitro and in preclinical mouse models of TNBC tumors. In extending these discoveries, we now show that aberrant YB1 expression activates oncogenic signaling leading to the dysregulation of cell cycle progression through the regulation of the Cyclin D/CDK4/6 complex signaling and the RB pathway. We also report the identification of a novel small molecule inhibitor, SU056, that specifically targets YB1 and inhibits its oncogenic activity in TNBC cell lines. We further show that the SU056-mediated inhibition of YB1, either as monotherapy or in combination with the CDK4/6 inhibitors, has a significant impact on inhibiting TNBC tumor progression and metastasis. In fact, the SU056-CDK4/6 inhibitors combination showed a synergistic effect when compared to monotherapy, at the same time reducing the toxicity associated with CDK4/6 inhibitors. Mechanistically, our studies revealed that genetic or pharmacologic targeting of YB1 inhibits TNBC tumor progression and metastasis through the regulation cyclin D-CDK4/6-RB pathway and blockade of cell cycle progression. Together, our data support the notion that targeting YB1 represents a potential therapeutic option for the treatment of TNBC, which could be enhanced when combined with standard of care treatment modalities. Citation Format: Wei Wang, Lamyae El Khalki, Neelum Yousaf Zai, Justin Szpendyk, Akram Alkrekshi, Bin Su, Khalid Sossey-Alaoui. YB1 is a novel therapeutic for the treatment of triple negative breast cancer tumors [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 5728.