Triple-negative Breast Cancer Cell Lines Research Articles

Significance of LIF/LIFR Signaling in the Progression of Obesity-Driven Triple-Negative Breast Cancer

American women with obesity have an increased incidence of triple-negative breast cancer (TNBC). The impact of obesity conditions on the tumor microenvironment is suspected to accelerate TNBC progression; however, the specific mechanism(s) remains elusive. This study explores the hypothesis that obesity upregulates leukemia inhibitory factor receptor (LIFR) oncogenic signaling in TNBC and assesses the efficacy of LIFR inhibition with EC359 in blocking TNBC progression. TNBC cell lines were co-cultured with human primary adipocytes, or adipocyte-conditioned medium, and treated with EC359. The effects of adiposity were measured using cell viability, colony formation, and invasion assays. Mechanistic studies utilized RNA-Seq, Western blotting, RT-qPCR, and reporter gene assays. The therapeutic potential of EC359 was tested using xenograft and patient-derived organoid (PDO) models. The results showed that adipose conditions increased TNBC cell proliferation and invasion, and these effects correlated with enhanced LIFR signaling. Accordingly, EC359 treatment reduced cell viability, colony formation, and invasion under adipose conditions and blocked adipose-mediated organoid growth and TNBC xenograft tumor growth. RNA-Seq analysis identified critical pathways modulated by LIF/LIFR signaling in diet-induced obesity mouse models. These findings suggest that adiposity contributes to TNBC progression via the activation of the LIF/LIFR pathway, and LIFR inhibition with EC359 represents a promising therapeutic approach for obesity-associated TNBC.

Synergistic Anti-Tumor Effects of Newcastle Disease Virus and Doxorubicin: Evidence from A Murine Breast Cancer Model

BackgroundDespite the recent advances in the diagnosis and treatment of breast cancer, triple-negative breast cancer (TNBC) remains a clinical challenge due to its aggressive nature and resistance to conventional therapies. Virotherapy has emerged as a promising cancer treatment strategy, leveraging the ability of viruses to specifically target and replicate in cancerous cells. This study evaluated the oncolytic potential of a combined therapeutic strategy, utilizing Newcastle disease virus (NDV) and Doxorubicin hydrochloride (Dox) both in vitro and in vivo. MethodsThe in vitro experiments involved exposing human and mouse TNBC cell lines (MDA-MB-231 and 4T1, respectively) to NDV and Dox, individually or in combination. Cell viability assays and flow cytometry analyses were conducted to assess the synergistic effects of NDV and Dox on regulating breast cancer cell behavior in vitro. Furthermore, the immune-stimulating potential of NDV was investigated by examining its effects on dendritic cell (DC) maturation using flow cytometry and T cell proliferation. The in vitro anti-tumor effects of NDV were examined in both parental and tamoxifen-resistant cancer cells to assess its efficacy against chemoresistance. Animal models of breast cancer were treated with NDV in combination with Dox. The body weight changes, tumor volume, and survival rates of the mice were monitored throughout the study. Histopathological analyses were conducted to evaluate the potential toxic effects of the treatments. ResultsBased on the MTT results, NDV at optimal concentrations synergized the effect of Dox to reduce the viability of both MDA-MB231 and 4T1 cell lines (Isobologram combination index of less than 1). Additionally, individual treatment with NDV was able to significantly reduce the viability of patient-derived breast cancer cells, compared to the untreated control (P < 0.05) without affecting the cells of normal adjacent tissue. Furthermore, a combination of NDV and Dox significantly enhanced the percentage of early and late apoptotic cells in MDA-MB-231 (P < 0.0001) and late apoptotic cells in 4T1 (P < 0.0001), in comparison with individual treatment with these agents. Flow cytometry results showed that, compared to wild type MDA-MB-231 cells, NDV-infected MDA-MB-231 cells were better inducers of T cell proliferation and DC maturation as evidenced by increased proliferation index (P < 0.05) and elevated expression of CD1a, CD83, and CD86 (P < 0.0001), respectively. Moreover, co-treatment of both wild-type and (tamoxifen) TAM-resistant MCF-7/TAMR-1 cells with TAM and NDV significantly reduce the viability of the cancer cells (P < 0.0001). In tumor-bearing mice locally engrafted with 4T1 cells, combined treatment of NDV and Dox exhibited a marked reduction in median tumor volume compared to the control group, validating our in vitro findings on their synergistic anti-tumor effects. These findings suggest that combining NDV with Dox can effectively inhibit tumor progression and has the potential to reduce the dose, and consequently the toxic side-effects, of Dox in breast cancer therapy.

FOS-Mediated PLCB1 Induces Radioresistance and Weakens the Antitumor Effects of CD8+ T Cells in Triple-Negative Breast Cancer.

Radioresistance and immune evasion are interactive and crucial events leading to treatment failure and progression of human malignancies. This research studies the role of phospholipase C beta 1 (PLCB1) in these events in triple-negative breast cancer (TNBC) and the regulatory mechanism. PLCB1 was bioinformatically predicted as a dysregulated gene potentially linked to radioresistance in TNBC. Parental TNBC cell lines were exposed to fractionated radiation for 6 weeks. PLCB1 expression was decreased in the first 2 weeks but gradually increased from Week 3. PLCB1 knockdown increased the radiosensitivity of the cells, as manifested by a decreased half-inhibitory dose of irradiation, reduced cell proliferation, apoptosis resistance, mobility, and tumorigenesis in mice. The FOS transcription factor promoted PLCB1 transcription and activated the PI3K/AKT signaling. Knockdown of FOS similarly reduced radioresistance and T cells-mediated immune evasion. However, the radiosensitivity of TNBC cells and the antitumor effects of CD8+ T cells could be affected by a PI3K/AKT activator or by the PLCB1 upregulation. The PLCB1 or FOS knockdown also suppressed radioresistance and tumorigenesis of the TNBC cells in mice. In conclusion, FOS-mediated PLCB1 induces radioresistance and weakens the antitumor effects of CD8+ T cells in TNBC by activating the PI3K/AKT signaling pathway.

Amine-Terminated Silver Nanoparticles Exhibit Potential for Selective Targeting of Triple-Negative Breast Cancer

Silver nanoparticles (AgNPs) demonstrate potential in treating aggressive cancers such as triple-negative breast cancer (TNBC) in preclinical models. To further the development of AgNP-based therapeutics for clinical use, it is essential to clearly define the specific physicochemical characteristics of the nanoparticles and connect these properties to biological outcomes. This study addresses this knowledge gap through detailed investigations into the structural and surface functional relationships, exploring the mechanisms, safety, and efficacy of AgNPs in targeting TNBC. The surface functionality of nanoparticles is crucial not only for their internalization into cancer cells but also for enhancing their toxicity toward tumor cells. Although the nanoparticles internalized into cancer cells, they failed to exhibit their full toxicity against the cancer. Herein we report a solvent-assisted synthesis amine, mercaptohexanol and bifunctional silver nanoparticles and performing comparative study to understand their selectivity and toxicity toward TNBC cells. The nanoparticles are fully characterized by UV–visible absorption spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and dynamic light scattering measurement (DLS). The synthesis method achieves an extremely high yield and surface coating ratio of synthesized colloidal AgNPs. Our findings reveal that the amine-capped AgNPs exhibit significant selective toxicity against TNBC cell lines MCF7 and MDA-MB-231 at a concentration of 40 µg/mL without affecting normal breast cell lines MCF10A. This study underscores the potential of functionalized AgNPs in developing safe and targeted therapeutic approaches for treating aggressive cancers like TNBC, laying the groundwork for future clinical advancements.

Mechanisms of Action of Sea Cucumber Triterpene Glycosides Cucumarioside A0-1 and Djakonovioside A Against Human Triple-Negative Breast Cancer.

Breast cancer is the most prevalent form of cancer in women worldwide. Triple-negative breast cancer is the most unfavorable for patients, but it is also the most sensitive to chemotherapy. Triterpene glycosides from sea cucumbers possess a high therapeutic potential as anticancer agents. This study aimed to identify the pathways triggered and regulated in MDA-MB-231 cells (triple-negative breast cancer cell line) by the glycosides cucumarioside A0-1 (Cuc A0-1) and djakonovioside A (Dj A), isolated from the sea cucumber Cucumaria djakonovi. Using flow cytometry, fluorescence microscopy, immunoblotting, and ELISA, the effects of micromolar concentrations of the compounds on cell cycle arrest, induction of apoptosis, the level of reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and expression of anti- and pro-apoptotic proteins were investigated. The glycosides caused cell cycle arrest, stimulated an increase in ROS production, and decreased Δψm in MDA-MB-231 cells. The depolarization of the mitochondrial membrane caused by cucumarioside A0-1 and djakonovioside A led to an increase in the levels of APAF-1 and cytochrome C. This, in turn, resulted in the activation of caspase-9 and caspase-3 and an increase in the level of their cleaved forms. Glycosides also affected the expression of Bax and Bcl-2 proteins, which are associated with mitochondria-mediated apoptosis in MDA-MB-231 cells. These results indicate that cucumarioside A0-1 and djakonovioside A activate the intrinsic apoptotic pathway in triple-negative breast cancer cells. Additionally, it was found that treatment with Cuc A0-1 resulted in in vivo inhibition of tumor growth and metastasis of murine solid Ehrlich adenocarcinoma.

Macropinocytosis mediates resistance to loss of glutamine transport in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) metabolism and cell growth uniquely rely on glutamine uptake by the transporter ASCT2. Despite previous data reporting cell growth inhibition after ASCT2 knockdown, we here show that ASCT2 CRISPR knockout is tolerated by TNBC cell lines. Despite the loss of a glutamine transporter and low rate of glutamine uptake, intracellular glutamine steady-state levels were increased in ASCT2 knockout compared to control cells. Proteomics analysis revealed upregulation of macropinocytosis, reduction in glutamine efflux and increased glutamine synthesis in ASCT2 knockout cells. Deletion of ASCT2 in the TNBC cell line HCC1806 induced a strong increase in macropinocytosis across five ASCT2 knockout clones, compared to a modest increase in ASCT2 knockdown. In contrast, ASCT2 knockout impaired cell proliferation in the non-macropinocytic HCC1569 breast cancer cells. These data identify macropinocytosis as a critical secondary glutamine acquisition pathway in TNBC and a novel resistance mechanism to strategies targeting glutamine uptake alone. Despite this adaptation, TNBC cells continue to rely on glutamine metabolism for their growth, providing a rationale for targeting of more downstream glutamine metabolism components.

Chemical Diversity of Aspergillus alliaceus Phenotypes: Discovery of Brominated Bianthrones with Activity against Triple-Negative Breast Cancer Cell Lines.

Marine-derived fungi have emerged as a source for novel metabolites with a broad range of bioactivities. However, accessing the full potential of fungi under standard laboratory conditions remains challenging. LC-MS-based metabolomics in combination with varied culture conditions is a fast and powerful tool to detect new metabolites. Here, three developmental forms of the marine-derived fungus Aspergillus alliaceus were analyzed and 14 fungal metabolites, including new brominated polyketides (11-14) were isolated. Structure elucidation relied mainly on 1D and 2D NMR techniques and was supported by low- and high-resolution mass spectrometry and DFT-based computations. We sequenced the A. alliaceus genome, identified the bianthrone-producing biosynthetic gene cluster, and conducted expression analysis on genes involved in sexual development and biosynthesis. The NCI-60 cell line panel revealed selective in vitro activity against triple-negative breast cancer (TNBC) for the halogenated allianthrones and their full antiproliferative and cytotoxic effects were evaluated in five TNBC cell lines.

GABA Type A receptors expressed in triple negative breast cancer cells mediate chloride ion flux.

Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset, limited unresponsiveness to hormone therapies and immunotherapy as well as high likelihood of metastasis and recurrence. Since no targeted standard treatment options are available for TNBC, novel and effective therapeutic targets are urgently needed. Ion channels have emerged as possible novel therapeutic candidates for cancer therapy. We previously showed that GABAA β3 subunit are expressed at higher levels in TNBC cell lines than non-tumorigenic MCF10A cells. GABAA β3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. However, it is not known if the upregulated GABAAR express at the cell-surface in TNBC and mediate Cl- flux. Cl- ions are known to play a role in cell-cycle progression in other cancers such as gastric cancer. Here, using surface biotinylation and (N-(Ethoxycarbonylmethyl)-6-Methoxyquinolinium Bromide) MQAE-dye based fluorescence quenching, we show that upregulated GABAAR are on the cell-surface in TNBC cell lines and mediate significantly higher chloride (Cl-) flux as compared to non-tumorigenic MCF10A cells. Moreover, this GABAAR mediated Cl- flux can be modulated by pharmacological agents and is decreased in TNBC cells with GABAA β3 subunit knockdown. Further, treatment of TNBC cells with bicuculline, a GABAAR antagonist reduced cell viability in TNBC cells Overall, these results point to an unexplored role of GABAAR mediated Cl- flux in TNBC.

BAFF overexpression in triple-negative breast cancer promotes tumor growth by inducing IL-10-secreting regulatory B cells that suppress anti-tumor T cell responses.

Despite BAFF's (B cell activating factor, BAFF) known influence on B cell survival and proliferation, its specific effects within the tumor microenvironment remain unclear. We aimed to elucidate how BAFF overexpression in breast cancer cells impacts tumor growth and the functions of T and B cells in the tumor microenvironment. BAFF was overexpressed in the 4T1 mouse triple-negative breast cancer cell line, and tumor growth, immune cell infiltration, and activity were assessed in vitro and in vivo using flow cytometry, co-culture assays, and mouse tumor models with B cell depletion. BAFF overexpression in 4T1 cells promoted tumor growth in vivo, suppressed CD8+ T cell activity, and increased IL-10-secreting CD5+ regulatory B cells in tumors. 4T1/BAFF cells directly enhanced IL-10 production in CD5+ B cells via BAFF/BAFF-receptor interactions, and IL-10 from CD5+ B cells inhibited IFN-γ secretion by T cells. B cell depletion partially reversed the tumor-promoting effects of BAFF overexpression. Our study reveals a novel mechanism by which BAFF can foster tumor progression, with the induction of IL-10-secreting regulatory B cells that suppress anti-tumor T cell responses appearing to be a key component of BAFF's tumor-promoting activity. These findings underscore the complex immunomodulatory effects that BAFF exerts in the tumor microenvironment and point to BAFF-induced regulatory B cells as a potential new therapeutic target in breast cancer that warrants further investigation.

Discovery of ZLC491 as a Potent, Selective, and Orally Bioavailable CDK12/13 PROTAC Degrader.

Selective degradation of cyclin-dependent kinases 12 and 13 (CDK12/13) emerges as a new potential therapeutic approach for triple-negative breast cancer (TNBC) and other human cancers. While several proteolysis-targeting chimera (PROTAC) degraders of CDK12/13 were reported, none are orally bioavailable. Here, we report the discovery of ZLC491 as a potent, selective, and orally bioavailable CDK12/13 PROTAC degrader. The compound effectively degraded CDK12 and CDK13 with DC50 values of 32 and 28 nM, respectively, in TNBC MDA-MB-231 cells. Global proteomic assessment and mechanistic studies revealed that ZLC491 selectively induced CDK12/13 degradation in a cereblon- and proteasome-dependent manner. Furthermore, the molecule efficiently suppressed transcription and expression of long genes, predominantly a subset of genes associated with DNA damage response, and significantly inhibited proliferation of multiple TNBC cell lines. Importantly, ZLC491 achieved an oral bioavailability of 46.8% in rats and demonstrated potent in vivo degradative effects on CDK12/13 in an MDA-MB-231 xenografted mouse model.

Nuclear localization of APLF facilitates breast cancer metastasis

Most breast cancer deaths result from metastases. We previously reported that DNA repair factor and histone chaperone Aprataxin PNK-like Factor (APLF) is involved in EMT-associated metastasis of triple negative breast cancer (TNBC) cells. However, non-metastatic cells also expressed APLF, the implications of which in disease advancement remain uncertain. Here, we demonstrate that the metastatic prognosis of breast cancer cells may be determined by the cellular localization of APLF. Using TNBC patient samples and cell lines, we discovered that APLF was localized in the nucleus and cytoplasm, whereas other subtypes of breast cancer had cytosolic or perinuclear localization. To investigate metastatic properties in vitro and in vivo, we modeled APLF differential localization by stably producing APLF-tagged nuclear localization signal (NLS) in the luminal subtype MCF7 cells in the absence of putative APLF NLS. Nuclear APLF in non-metastatic MCF7 cells demonstrated pronounced migration, invasion and metastatic potential. We obtained the mechanistic insight from molecular studies that PARP1 could facilitate the transport of APLF from the cytosol to the nucleus, assisting in the metastasis of TNBC cells linked with EMT. Inhibition of PARP1 enzymatic activity with olaparib abrogated the nuclear expression of APLF with loss in expression of genes associated with EMT. Thus, our findings reveal that cellular localization of APLF may predict the risk of breast cancer to metastasize and hence could be exploited to determine the disease progression. We anticipate that the inhibition of cytosolic PARP1-APLF interaction may potentially aid in the prevention of breast cancer metastasis in TNBC patients.

An engineered model of metastatic colonization of human bone marrow reveals breast cancer cell remodeling of the hematopoietic niche

Incomplete understanding of metastatic disease mechanisms continues to hinder effective treatment of cancer. Despite remarkable advancements toward the identification of druggable targets, treatment options for patients in remission following primary tumor resection remain limited. Bioengineered human tissue models of metastatic sites capable of recreating the physiologically relevant milieu of metastatic colonization may strengthen our grasp of cancer progression and contribute to the development of effective therapeutic strategies. We report the use of an engineered tissue model of human bone marrow (eBM) to identify microenvironmental cues regulating cancer cell proliferation and to investigate how triple-negative breast cancer (TNBC) cell lines influence hematopoiesis. Notably, individual stromal components of the bone marrow niche (osteoblasts, endothelial cells, and mesenchymal stem/stromal cells) were each critical for regulating tumor cell quiescence and proliferation in the three-dimensional eBM niche. We found that hematopoietic stem and progenitor cells (HSPCs) impacted TNBC cell growth and responded to cancer cell presence with a shift of HSPCs (CD34+CD38-) to downstream myeloid lineages (CD11b+CD14+). To account for tumor heterogeneity and show proof-of-concept ability for patient-specific studies, we demonstrate that patient-derived tumor organoids survive and proliferate in the eBM, resulting in distinct shifts in myelopoiesis that are similar to those observed for aggressively metastatic cell lines. We envision that this human tissue model will facilitate studies of niche-specific metastatic progression and individualized responses to treatment.

Targeting oncogenic MAGEA6 sensitizes triple negative breast cancer to doxorubicin through its autophagy and ferroptosis by stabling AMPKα1

Melanoma-associated antigen A6 (MAGEA6) is well known to have oncogenic activity, but the underlying mechanisms by which it regulates tumor progression and chemo-resistance, especially in triple-negative breast cancer (TNBC), have been unknown. In the study, the differential expression genes (DEGs) in TNBC tumor tissues and TNBC-resistant tumor tissues were analyzed based on TCGA and GEO datasets. MAGEA6, as the most significantly expressed gene, was analyzed by RT-qPCR, western blotting and immunohistochemistry assay in TNBC cell lines and tumor tissues. The potential mechanisms that influence chemo-resistance were also evaluated. Results displayed that MAGEA6 was highly expressed in TNBC and involved in drug resistance. MAGEA6 silencing enhanced the chemo-sensitivity of TNBC to doxorubicin (DOX) in vitro and in vivo, as determined by decreasing IC50 value, proliferation and invasion capacity, and triggering apoptosis. Mechanistically, it was shown that MAGEA6 depletion sensitized TNBC to DOX via regulating autophagy. Ubiquitination assay displayed that knockdown of MAGEA6 decreased the AMPKα1 ubiquitination, thereby elevating the levels of AMPKα1 and p-AMPKα in TNBC cells. Importantly, AMPK inhibitor (Compound C) can reduce the LC3II/I level induced by sh-MAGEA6, indicating that sh-MAGEA6 activated AMPK signaling through suppressing AMPKα1 ubiquitination and then facilitated autophagy in TNBC. Furthermore, we also observed that AMPK is required for SLC7A11 to regulate ferroptosis, and supported the crux roles of MAGEA6/AMPK/SLC7A11-mediated ferroptosis on modulating DOX sensitivity in TNBC cells. These findings indicated that targeting MAGEA6 can enhance the chemo-sensitivity in TNBC via activation of autophagy and ferroptosis; its mechanism involves AMPKα1-dependent autophagy and AMPKα1/SLC7A11-induced ferroptosis.

8743 Regulation Of Tryptophan 2,3-Dioxygenase 2 And Androgen Receptor In Triple-Negative Breast Cancer

Abstract Disclosure: L. Kuo: None. K. Lacey: None. N. Spoelstra: None. J.K. Richer: None. Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype with a peak recurrence rate within 3-5 years after initial diagnosis. Androgen receptor (AR) and Tryptophan 2,3-dioxygenase 2 (TDO2) are elevated in TNBC cells grown under anchorage-independent conditions and facilitate anoikis resistance. Several studies reported that the luminal AR subset of TNBC showed a positive enrichment of the tryptophan (TRP) catabolism pathway gene signature. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) inhibition decreases AR promoter transcriptional activity in prostate cancer. We have observed that TNBC with high AR have high levels of genes/proteins in the tryptophan catabolism pathway including the rate limiting enzyme TDO2. Hypothesis: TDO2 activity regulates AR expression through the downstream tryptophan catabolite kynurenine (KYN), which binds and activates AhR and AhR and AR may work together to promote TNBC progression. Methods: Human TNBC cell lines: MDA-MB-453, BT549, SUM159PT ​ were used in this study and the expression of TDO2 and AR were examined by immunohistochemistry (IHC) and western blot. TDO2 was stably knocked down (KD) via shRNA or overexpressed (OE) using lentivirus transduction. Mass spectrometry-based metabolomics were used to measure changes in TRP catabolite levels, and a cytokine array (R&amp;D) was utilized to analyze conditioned media. Results: TDO2 and AR protein are positively correlated in human TNBC cell lines. Although overexpression of TDO2 did not alter AR protein expression in SUM159PT, in the human MDA-MB-453, which have high levels of AR, knockdown of TDO2 (shTDO2) reduced AR protein by 40% and production of kynurenine (KYN) was reduced by 50% (p&amp;lt;0.0001). This suggests that TDO2 activity, specifically KYN binding to AhR, may influence AR protein levels. Conditioned media (CM) from TDO2 OE and KD respectively increased and decreased CXCL5 and IGFBP2. Lastly, TCGA database analysis of TNBC tumors with high levels of both AhR and AR showed significantly shorter patient survival (HR=1.47, log rank p=0.046) compared to those with tumors with lower amounts of both genes. Conclusion: TDO2 and AR are positively correlated in human TNBC cell lines. Tumors with high levels of both AR and AhR result in significantly shorter survival time for TNBC patients. Knockdown of TDO2 significantly reduced KYN production and AR expression in luminal AR TNBC cell lines and future experiments will focus on the underlying mechanism behind this and whether it is dependent on KYN-mediated activation of AhR and if AhR stabilizes AR in TNBC. Presentation: 6/3/2024

8743 Regulation of Tryptophan 2,3-Dioxygenase 2 and Androgen Receptor in Triple-Negative Breast Cancer

Abstract Disclosure: L. Kuo: None. K. Lacey: None. N. Spoelstra: None. J.K. Richer: None. Background: Triple Negative Breast Cancer (TNBC) is an aggressive subtype with a peak recurrence rate within 3-5 years after initial diagnosis. Androgen receptor (AR) and Tryptophan 2,3-dioxygenase 2 (TDO2) are elevated in TNBC cells grown under anchorage-independent conditions and facilitate anoikis resistance. Several studies reported that the luminal AR subset of TNBC showed a positive enrichment of the tryptophan (TRP) catabolism pathway gene signature. Recent studies have demonstrated that aryl hydrocarbon receptor (AhR) inhibition decreases AR promoter transcriptional activity in prostate cancer. We have observed that TNBC with high AR have high levels of genes/proteins in the tryptophan catabolism pathway including the rate limiting enzyme TDO2. Hypothesis: TDO2 activity regulates AR expression through the downstream tryptophan catabolite kynurenine (KYN), which binds and activates AhR and AhR and AR may work together to promote TNBC progression. Methods: Human TNBC cell lines: MDA-MB-453, BT549, SUM159PT ​ were used in this study and the expression of TDO2 and AR were examined by immunohistochemistry (IHC) and western blot. TDO2 was stably knocked down (KD) via shRNA or overexpressed (OE) using lentivirus transduction. Mass spectrometry-based metabolomics were used to measure changes in TRP catabolite levels, and a cytokine array (R&amp;D) was utilized to analyze conditioned media. Results: TDO2 and AR protein are positively correlated in human TNBC cell lines. Although overexpression of TDO2 did not alter AR protein expression in SUM159PT, in the human MDA-MB-453, which have high levels of AR, knockdown of TDO2 (shTDO2) reduced AR protein by 40% and production of kynurenine (KYN) was reduced by 50% (p&amp;lt;0.0001). This suggests that TDO2 activity, specifically KYN binding to AhR, may influence AR protein levels. Conditioned media (CM) from TDO2 OE and KD respectively increased and decreased CXCL5 and IGFBP2. Lastly, TCGA database analysis of TNBC tumors with high levels of both AhR and AR showed significantly shorter patient survival (HR=1.47, log rank p=0.046) compared to those with tumors with lower amounts of both genes. Conclusion: TDO2 and AR are positively correlated in human TNBC cell lines. Tumors with high levels of both AR and AhR result in significantly shorter survival time for TNBC patients. Knockdown of TDO2 significantly reduced KYN production and AR expression in luminal AR TNBC cell lines and future experiments will focus on the underlying mechanism behind this and whether it is dependent on KYN-mediated activation of AhR and if AhR stabilizes AR in TNBC. Presentation: 6/3/2024

7076 Inhibiting Oxidosqualene Cyclase, An Enzyme In The Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis Of Triple-Negative Breast Cancer Cells

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 105 cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 105 MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p&amp;lt;0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024

12347 Cholesterol Biosynthesis Inhibitor Ro 48-8071 Suppresses Growth Of Triple-negative Breast Cancer Cells By Inducing Apoptosis And Suppressing Angiogenesis

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu, proteins that are commonly targeted in breast cancer therapies. Women who suffer from TNBC have limited treatment options and are administered toxic chemotherapeutic agents. They have poor prognosis due to the emergence of drug-resistant cells that lead to tumor metastasis. New non-toxic therapeutic strategies to control TNBC progression, and prevent metastasis are urgently needed. Cholesterol is an essential structural and functional component of cell membranes necessary for tumor growth and progression. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy since tumor cells grow rapidly, and need a greater supply of cholesterol. We targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor growth. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors but its effect on TNBC is not known. Pharmacological doses of RO potently reduced the viability of both MDA-MB-231 and BT20 TNBC cells in a dose dependent manner (1-50 μM). IC50 values for loss of cell viability was approximately 10 μM for both cell lines following 48 h exposure to RO. Doses close to the IC50 for OSC (1-10 nM) also reduced cell viability following exposure of TNBC cells to RO for a week. Importantly, RO had no effect on the viability of normal human mammary cells. FACS analysis revealed that RO dose-dependently induced apoptosis of TNBC cell lines in vitro. In vivo, administration of 5-10 mg/kg RO to mice with BT-20 TNBC cell-derived xenografts (100 mm3) inhibited tumor growth with no apparent toxicity. Immunohistochemical analysis of tumor tissues at the end of treatment showed that RO increased TUNEL expression, suggesting that the in vivo effects of RO were also exerted through mechanisms affecting apoptosis. Furthermore, IHC of tumor sections showed that RO also reduced levels of the angiogenic markers VEGF and CD-31 (blood vessels) in vivo, indicating that suppression of angiogenesis is also part of the mechanism through which RO exerts its anti-tumor effects. We conclude that RO is a potent inhibitor of TNBC and that its anti-tumor effects involve mechanisms that induce apoptosis and suppress angiogenesis. Presentation: 6/2/2024

7076 Inhibiting Oxidosqualene Cyclase, an Enzyme in the Cholesterol Biosynthetic Pathway, Suppresses Lung Metastasis of Triple-negative Breast Cancer Cells

Abstract Disclosure: Y. Liang: None. S.M. Hyder: None. Triple-negative breast cancers (TNBC) lack ER, PR and Her2neu proteins that are commonly targeted in breast cancer therapies. As a consequence, women who suffer from TNBC have poor prognosis and limited treatment options, and are usually administered toxic, non-specific chemotherapeutic agents. Drug-resistance almost always occurs, leading to tumor metastasis, the main cause of patient death. New non-toxic therapeutic strategies to control metastasis of TNBC are urgently needed. Cholesterol is an essential structural and functional component of cell membranes that is necessary for the growth of both primary tumors and metastatic colonies that reside in distant organs. Therefore, inhibiting cholesterol production is an attractive therapeutic strategy. Treatment with statins, a class of cholesterol biosynthesis inhibitors that target HMGCoA-reductase, is associated with certain undesirable side effects; consequently, we targeted oxidosqualene cyclase (OSC), an enzyme that occurs downstream of HMGCoA-reductase in the cholesterol biosynthetic pathway, to disrupt tumor metastasis. Potent small molecule inhibitors of OSC have been identified. RO 48-8071 (4′-[6-(allylmethylamino)hexyloxy]-4-bromo-2′-fluorobenzophenone fumarate) (RO), has emerged as a useful chemotherapeutic agent for treating several forms of primary tumors. We developed a mouse model for studying lung metastasis using a MDA-MB-231 variant (LM2) TNBC cell line obtained from Dr. Massagué (Memorial Sloan-Kettering Cancer Center). These are aggressive cells, and injection of only 2 X 10[5] cells produces detectable lung metastatic colonies in approximately 4-6 weeks. We inoculated female nu/nu mice, 5-6 weeks old, with 2 X 10[5] MDA-MB-231/LM2 cells in 0.1 ml DMEM/F12 medium without serum via tail vein. Five days after tumor cell inoculation animals received 20 mg/kg (n=6), 40 mg/kg (n=7) RO (ip) or vehicle alone (n=7) every other day for two weeks. Animals were then sacrificed, and both lungs harvested and immediately fixed in Bouin’s fixative. Metastatic colonies on the lung surface were identified and counted under a dissecting microscope. Our study showed that RO effectively reduced lung metastasis without affecting animal weight. Compared with 56 + 7 colonies (Mean ± SEM) in controls given vehicle alone, RO significantly reduced lung metastatic colonies to 28 ± 5 in animals exposed to 20 mg/kg and 6 ± 1 in 40 mg/kg treated animals (p&amp;lt;0.05, ANOVA). Reduction in number of colonies in 40 mg/kg group was also significantly different from colonies counted in 20 mg/kg treated animals. Our study is the first to demonstrate that disruption of cholesterol biosynthesis in a model of human TNBC leads to reduced metastasis in lungs. Further studies will determine mechanisms through which RO suppresses TNBC metastasis to the lungs. Supported by Zalk Missouri Professor Endowment Funds. Presentation: 6/3/2024

Citrus wastewater as a source of value-added products: Quali-quantitative analysis and in vitro screening on breast cancer cell lines.

Citrus wastewater from industries is a source of bioactive compounds whose recovery could be a useful approach to convert processing waste into potential resources to be exploited in food, pharmaceutical, and chemical companies. Citrus wastewater, obtained from the industrial processing of Citrus sinensis, was freeze-dried and qualitative/quantitative evaluated using HPLC/MS Q-TOF analysis. Antiproliferative activity was investigated on MDA-MB-231 (triple-negative breast cancer cell line), MCF-7 (breast cancer cell line), and its multidrug-resistant variant MCF-7R. Fraction 8 emerged for its cytotoxicity toward MCF-7R cells. Its main component, the polymethoxylated flavone nobiletin (80%), is likely involved in increasing the number of G1-phase MCF-7R cells without inducing cell death. Notably, fraction 8 sensitizes MCF7-R cells to the antiproliferative effects of doxorubicin, thus contributing to overcoming MCF7-R multidrug resistance. Our studies highlighted the possibility of applying a sustainable strategy for citrus wastewater recycling to recover functional compounds as useful adjuvants for the prevention and treatment of malignancies.

Single-cell transcriptional atlas of human breast cancers and model systems.

Breast cancer's complex transcriptional landscape requires an improved understanding of cellular diversity to identify effective treatments. The study of genetic variations among breast cancer subtypes at single-cell resolution has potential to deepen our insights into cancer progression. In this study, we amalgamate single-cell RNA sequencing data from patient tumours and matched lymph metastasis, reduction mammoplasties, breast cancer patient-derived xenografts (PDXs), PDX-derived organoids (PDXOs), and cell lines resulting in a diverse dataset of 117 samples with 506719 total cells. These samples encompass hormone receptor positive (HR+), human epidermal growth factor receptor 2 positive (HER2+), and triple-negative breast cancer (TNBC) subtypes, including isogenic model pairs. Herein, we delineated similarities and distinctions across models and patient samples and explore therapeutic drug efficacy based on subtype proportions. PDX models more closely resemble patient samples in terms of tumour heterogeneity and cell cycle characteristics when compared with TNBC cell lines. Acquired drug resistance was associated with an increase in basal-like cell proportions within TNBC PDX tumours as defined with SCSubtype and TNBCtype cell typing predictors. All patient samples contained a mixture of subtypes; compared to primary tumours HR+ lymph node metastases had lower proportions of HER2-Enriched cells. PDXOs exhibited differences in metabolic-related transcripts compared to PDX tumours. Correlative analyses of cytotoxic drugs on PDX cells identified therapeutic efficacy was based on subtype proportion. We present a substantial multimodel dataset, a dynamic approach to cell-wise sample annotation, and a comprehensive interrogation of models within systems of human breast cancer. This analysis and reference will facilitate informed decision-making in preclinical research and therapeutic development through its elucidation of model limitations, subtype-specific insights and novel targetable pathways. Patient-derived xenografts models more closely resemble patient samples in tumour heterogeneity and cell cycle characteristics when compared with cell lines. 3D organoid models exhibit differences in metabolic profiles compared to their in vivo counterparts. A valuable multimodel reference dataset that can be useful in elucidating model differences and novel targetable pathways.