Effects In Triple-negative Breast Cancer Research Articles

Coptisine exerts anti-tumour effects in triple-negative breast cancer by targeting mitochondrial complex I.

Triple-negative breast cancer (TNBC) has a poor prognosis due to limited therapeutic options. Recent studies have shown that TNBC is highly dependent on mitochondrial oxidative phosphorylation. The aim of this study was to investigate the potential of coptisine, a novel compound that inhibits the complex I of the mitochondrial electron transport chain (ETC), as a treatment for TNBC. In this study, mitochondrial metabolism in TNBC was analysed by bioinformatics. In vitro and in vivo experiments (in mice) were conducted to evaluate the potential of coptisine as an ETC complex I-targeting therapeutic agent and to investigate the molecular mechanisms underlying coptisine-induced mitochondrial dysfunction. The therapeutic effect of coptisine was assessed in TNBC cells and xenograft mouse model. We demonstrated that mitochondrial ETC I was responsible for this metabolic vulnerability in TNBC. Furthermore, a naturally occurring compound, coptisine, exhibited specific inhibitory activity against this complex I. Treatment with coptisine significantly inhibited mitochondrial functions, reprogrammed cellular metabolism, induced apoptosis and ultimately inhibited the proliferation of TNBC cells. Additionally, coptisine administration induced prominent growth inhibition that was dependent on the presence of a functional complex I in xenograft mouse models. Altogether, these findings suggest the promising potential of coptisine as a potent ETC complex I inhibitor to target the metabolic vulnerability of TNBC.

Abstract PO4-06-10: Targeting necroptosis in breast cancer induced by Ophiobolin A

Abstract Abstract Necroptosis or programmed necrosis is a form of cell death distinct from apoptosis. Necroptotic cells display the character of rounding of the cell, gain in cell volume, rupture of plasma membrane and release of intra-cellular contents. Although few key necroptosis regulators including receptor interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain like pseudokinase (MLKL) has been identified, their role/mechanism in inducing cell death in triple-negative breast cancer (TNBC) which lack RIPK3 expression remains elusive. Ophiobolin A is a compound naturally made by fungus and potently kills cancer cells especially TNBC. Our current study investigated the cell death mechanism(s) induced by Ophiobolin A (natural product) in breast cancer cell lines. The mechanism of cell death elicited by OpA was analyzed by applying inhibitors for specific pathways including apoptosis, necroptosis, ferroptosis and paraptosis, by analyzing markers of cell death using flow cytometry and western blotting. The formation of necrosomes was analyzed with exogeneous expression of RIPK3 tagged with green fluorescence protein (GFP) in HeLa, MDA-MB-231, MDA-MB-468 cell lines which lacks endogenous RIPK3 expression and in MCF7 estrogen receptor (ER) positive cell line. OpA induced necrosome formation was identified by formation of puncta in TNBC and HeLa cells which lack RIP3 expression, however, necrosomes were not detectable in ER positive cells. Clinical relevance of RIPK3 and MLKL has been analyzed using bioinformatic tools. Effect of OpA In vivo has been analyzed using Immune compromised SCID mice. The data obtained in the present study indicates that OpA has an increased anticancer effect in TNBC compared to the estrogen receptor (ER) positive breast cancer cells. Cells having undergone EMT were shown to have more sensitivity towards OpA when compared to uninduced cell lines, which is sensitive to Necrostatin-1, an inhibitor of necroptosis. However, apoptosis, ferroptosis and paraptosis inhibitors failed to rescue cell death which clearly confirms that OpA induces necroptosis cell death pathway in EMT undergone cells. OpA treatment significantly increased puncta in TNBC compared non-TNBC cells, further confirming the necroptotic cell death in TNBC. Bioinformatic analysis revealed that RIPK3 is negatively corelated with breast cancer and has least over-all survival in patients who lack RIPK3 expression. OpA treatment showed a significant tumor regression in tumor bearing SCID mice. All together our findings identify that OpA induce necroptosis in EMT-enriched TNBC. The study further needs an in-depth analysis of molecular mechanism induced by OpA and involvement of EMT in necroptotic cell death. Citation Format: Tolulope Ojo, Santhalakshmi Ranganathan. Targeting necroptosis in breast cancer induced by Ophiobolin A [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 PO4-06-10.

Abstract PO4-18-07: A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in triple negative breast cancer and transcriptionally addicted relapsed or refractory solid tumors

Abstract Background: MYC deregulation is a hallmark of triple negative breast cancer (TNBC) and is associated with aggressive tumors and poor clinical outcomes. Although MYC remains undrugged, targeting of its cofactors has emerged as an attractive strategy to inhibit MYC oncogenic activity. Cyclin-dependent kinase 9 (CDK9) is a critical regulator of oncogenic MYC expression and an important MYC cofactor. KB-0742 is an oral CDK9 inhibitor that demonstrates promising preclinical activity against TNBC. In a real-world cohort, TNBCs have higher MYC expression and higher rates of MYC genomic amplification than other breast cancer subtypes. In primary patient-derived cell lines, KB-0742 treatment results in stronger cytotoxic effects in TNBC as compared to other subtypes. In patient-derived organoids and patient-derived xenografts, CDK9 inhibition by KB-0742 drives antiproliferative and anti-tumor growth effects, including in models that are resistant to standard of care. KB-0742 strongly downregulates MYC protein levels at doses that only partially inhibit CDK9 activity, suggesting a therapeutic window for tumor-specific activity (Saffran, D.C. et al, 2021). KB-0742 is currently being evaluated in a phase 1/2 dose escalation and cohort expansion study in patients with TNBC and other transcriptionally addicted tumors (NCT04718675). Trial design: This phase 1/2 study includes two parts: dose escalation (part 1) and cohort expansion (part 2). Part 2 includes cohort A (solid tumors with high prevalence of MYC overexpression including TNBC, non-small cell lung cancer and ovarian) and cohort B (other transcriptionally addicted tumor types including sarcomas, adenoid cystic carcinoma, nut midline carcinoma and small cell lung cancer). KB-0742 is dosed orally once daily for 3 consecutive days, followed by 4 days, off on a weekly basis in 28-day cycles until unacceptable toxicity or disease progression. Eligibility criteria: Part 1 dose escalation is open to patients with relapsed or refractory solid tumors. Part 2 is defined by tumor indications in cohorts A and B. Eligibly criteria include age > 18 years (≥ 12 years old and with a body weight ≥ 40 kg part 2 for cohort B), acceptable organ function, and ECOG PS < 2. Specific aims: Primary objectives include evaluation of pharmacokinetics (PK), pharmacodynamics (PD), safety, tolerability, preliminary anti-tumor activity, and identifying a maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). PK measurements include Cmax, tmax, AUC0-last, accumulation ratio (Racc) and t1/2. Safety data will be evaluated per the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI-CTCAE) v5.0. The Modified Continuous Reassessment Method (mCRM) (Goodman et al., Stat Med 1995) will guide dose escalation and MTD. RP2D nomination is informed by PD in peripheral blood mononuclear cells using assays to evaluate phosphorylation of the CDK9 substrate serine 2 on the RNA Polymerase II C-terminal domain (pSER2) and CDK9-responsive gene expression. Radiographic tumor response to KB-0742 in patients is assessed every other cycle starting from cycle two after treatment using RECIST 1.1 criteria. Target accrual: Targeted total enrollment is 170 patients. For additional information, contact clinicaltrials@kronosbio.com Citation Format: Monica Mita, Alain Mita, Miguel Villalona-Calero, Noah Federman, Drew Rasco, David Spigel, Jia Luo, Gregory Cote, Richard Cutler, Pavan Kumar, Crystal MacKenzie, Charles Lin, Jorge DiMartino, Elizabeth Olek, Brian Van Tine. A dose escalation and cohort expansion study of the CDK9 inhibitor KB-0742 in triple negative breast cancer and transcriptionally addicted relapsed or refractory solid tumors [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 PO4-18-07.

Abstract 7459: iNOS inhibition through L-NMMA reveals immunostimulatory effects in obesity-associated triple-negative breast cancer (TNBC) tumors

Abstract Purpose of study: This study examines how obesity, an inflammatory condition, impacts treatment outcomes in triple-negative breast cancer (TNBC). Obesity fosters an unfavorable tumor microenvironment (TME) marked by immune suppression, metabolic shifts, and protumoral cytokine release. Elevated nitric oxide (NO) levels linked with obesity compromise vascular integrity, promoting breast cancer metastasis. This study examines whether combining standard chemotherapy (docetaxel) with NOS inhibition via L-NMMA could reshape the TME and bolster antitumor effects in TNBC within an obese mouse model. Experimental Procedure: C57bl/6 mice, a TNBC syngeneic mouse model, were fed a high-fat diet (HFD) or normal diet (ND) for 10 weeks. Tumors were induced orthotopically via E0771 cell line injection into the mammary fat pad. Upon reaching 100 mm^3, mice were randomized into vehicle and L-NMMA treatment groups. Digital spatial transcriptomics using NanoString™ GeoMx and nCounter platforms were conducted on FFPE tumor tissue sections from ND, HFD, and HFD+LNMMA treated mice. A panel targeting 64 protein markers aided in immune cell phenotyping and TME assessment. Results: HFD-induced tumors exhibited increased growth, elevated pro-inflammatory cytokine levels, and heightened iNOS expression. Unsupervised hierarchical clustering after nCounter analysis revealed a distinct protein expression signature in HFD tumors associated with T cell infiltration and exhaustion, as evidenced by the upregulation of CD3, CD4, PD-1/PD-L1, FOXP3, and CTLA 4. Markers for identifying immunosuppressed macrophages (F4/80 and CD163) were also elevated. Remarkably, LNMMA treatment on HFD tumors led to reduced tumor growth and lower expression levels of these markers, alongside a downregulation of survival/proliferation markers such as p53, Ki67, and key members of the Pi3k signaling pathway such as phospoAktSer473. Conclusions: Our findings indicate a distinct immunomodulatory mechanism of iNOS inhibition in HFD tumors, which displayed expression profiles marked by heightened immunosuppressive markers when compared to ND tumors. LNMMA treatment reversed this phenotype, aligning the profile closer to ND tumors. These discoveries underscore the potential of inhibiting iNOS to reshape the immune tumor microenvironment (TME) and modulate pathways linked to cell proliferation and survival. This presents a promising avenue for enhancing the effectiveness of treatments for triple-negative breast cancer (TNBC). Citation Format: Ivonne Uzair, Kai Sun, Seun Hynes, Wei Qian, Jianying Zhou, Karina Ortega Martinez, Liliana Guzman, Polina Matre, Jenny Chang. iNOS inhibition through L-NMMA reveals immunostimulatory effects in obesity-associated triple-negative breast cancer (TNBC) 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 7459.

Abstract 3171: The effect of estrogen and phytoestrogens in Inflammatory breast cancer

Abstract Inflammatory breast cancer is an aggressive and lethal type of breast cancer. Frequently, this cancer is diagnosed in an advanced stage with metastasis. IBC diagnosis is associated with a worse survival rate than other types of breast cancer. The presence of dermal and stromal tumor emboli blocking the lymphatic vessels under the skin is considered a hallmark of IBC and assumed to be responsible for the high metastatic behavior and aggressiveness of IBC disease. To this date, there are no effective targeted therapeutics, especially for those patients that account for approximately 20-40% of IBC cases with triple-negative breast cancer (TNBC) classification. Several studies have shown that estrogen can exert non-genomic effects in IBC and non-IBC TNBC, mediated by the expression of alternate estrogen receptors including estrogen receptor alpha-36, and GPR30. In this context, estrogen can activate non-genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, naturally derived structural analogs to 17β-estradiol (E2), were evaluated as an alternative treatment option using in vitro IBC cell models (SUM149 and SUM190). Given the estrogenic properties of phytoestrogens, the drugs were hypothesized to interact with targets in pathways similar to E2 in non-genomic signaling. Several phytoestrogens including coumestrol (Cou) can induce a cytotoxic effect in TNBC. We hypothesize (1) that estrogen non-genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non-genomic signaling in IBC. To test these hypotheses, IBC cells were treated with E2 or Cou to determine their effects on the activation of downstream kinases by western blot and phospho-kinase array assays. In addition, transcriptional effects upon treatment with Cou were identified using RNA-seq. A dose-response curve in 2D and 3D models was generated to determine the half-maximal inhibitory concentration (IC50) of Cou. Finally, treatment using the IC50 of Cou caused a decrease in cell viability in the TNBC cell line. Also, in comparison to E2, Cou decreases migration, proliferation, and tumor emboli formation in IBC cell lines. In addition, IBC cells were treated using monotherapy and combination therapy with phytoestrogens including Cou, genistein (GN), and/or 8-prenylnaringenin (8-PN). Observed reductions of cell viability in IBC cell lines treated with the different phytoestrogens and combinations make them worthy of further characterization as anti-cancer therapies in in vitro and in vivo models. In summary, IBC cells are responsive to E2 making this signaling pathway attractive for the development of innovative therapeutic strategies with phytoestrogens as potential candidates for treatment. Citation Format: Esther A. Peterson-Peguero, Keishla Rodriguez-Martir, Madeline Ganshert, Dalissa Negron-Figueroa, Ayeisha N. Colon-Ortiz. The effect of estrogen and phytoestrogens in Inflammatory 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 3171.

20(R)-Panaxatriol enhances METTL3-mediated m6A modification of STUB1 to inhibit autophagy and exert antitumor effects in Triple-Negative Breast Cancer cells

BackgroundAberrant activation of autophagy in triple-negative breast cancer (TNBC) has led researchers to investigate potential therapeutic strategies targeting this process. The regulation of autophagy is significantly influenced by METTL3. Our previous research has shown that the Panax ginseng-derived compound, 20(R)-panaxatriol (PT), has potential as an anti-tumor agent.However, it remains unclear whether PT can modulate autophagy through METTL3 to exert its anti-tumor effects. ObjectiveOur objective is to investigate whether PT can regulate autophagy in TNBC cells and elucidate the molecular mechanisms. Study DesignFor in vitro experiments, we employed SUM-159-PT and MDA-MB-231 cells. While in vivo experiments involved BALB/c nude mice and NOD/SCID mice. MethodsIn vitro, TNBC cells were treated with PT, and cell lines with varying expression levels of METTL3 were established. We assessed the impact on tumor cell activity and autophagy by analyzing autophagic flux, Western Blot (WB), and methylation levels. In vivo, subcutaneous transplantation models were established in BALB/c nude and NOD/SCID mice to observe the effect of PT on TNBC growth. HE staining and immunofluorescence were employed to analyze histopathological changes in tumor tissues. MeRIP-seq and dual-luciferase reporter gene assays were used to identify key downstream targets. Additionally, the silencing of STIP1 Homology And U-Box Containing Protein 1 (STUB1) explored PT's effects. The mechanism of PT's action on STUB1 via METTL3 was elucidated through mRNA stability assays, mRNA alternative splicing analysis, and nuclear-cytoplasmic mRNA separation. ResultsIn both in vivo and in vitro experiments, it was discovered that PT significantly upregulates the expression of METTL3, leading to autophagy inhibition and therapeutic effects in TNBC. Simultaneously, through MeRIP-seq analysis and dual-luciferase reporter gene assays, we have demonstrated that PT modulates STUB1 via METTL3, influencing autophagy in TNBC cells. Furthermore, intriguingly, PT extends the half-life of STUB1 mRNA by enhancing its methylation modification, thereby enhancing its stability. ConclusionIn summary, our research reveals that PT increases STUB1 m6A modification through a METTL3-mediated mechanism in TNBC cells, inhibiting autophagy and further accentuating its anti-tumor properties. Our study provides novel mechanistic insights into TNBC pathogenesis and potential drug targets for TNBC.

Precise nano-system-based drug delivery and synergistic therapy against androgen receptor-positive triple-negative breast cancer

Targeting androgen receptor (AR) has shown great therapeutic potential in triple-negative breast cancer (TNBC), yet its efficacy remains unsatisfactory. Here, we aimed to identify promising targeted agents that synergize with enzalutamide, a second-generation AR inhibitor, in TNBC. By using a strategy for screening drug combinations based on the Sensitivity Index (SI), we found that MK-8776, a selective checkpoint kinase1 (CHK1) inhibitor, showed favorable synergism with enzalutamide in AR-positive TNBC. The combination of enzalutamide and MK-8776 was found to exert more significant anti-tumor effects in TNBC than the single application of enzalutamide or MK-8776, respectively. Furthermore, a nanoparticle-based on hyaluronic acid (HA)-modified hollow-manganese dioxide (HMnO2), named HMnE&M@H, was established to encapsulate and deliver enzalutamide and MK-8776. This HA-modified nanosystem managed targeted activation via pH/glutathione responsiveness. HMnE&M@H repressed tumor growth more obviously than the simple addition of enzalutamide and MK-8776 without a carrier. Collectively, our study elucidated the synergy of enzalutamide and MK-8776 in TNBC and developed a novel tumor-targeted nano drug delivery system HMnE&M@H, providing a potential therapeutic approach for the treatment of TNBC.

Current progress and prospects for G protein-coupled estrogen receptor in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a biologically and clinically heterogeneous disease. The G protein-coupled estrogen receptor (GPER) plays a crucial role in mediating the effect of estrogen and estrogen-like compounds in TNBC cells. Compared with other subtypes, GPER has a higher expression in TNBC. The GPER mechanisms have been thoroughly characterized and analyzed in estrogen receptor α (ERα) positive breast cancer, but not in TNBC. Our previous work revealed that a higher expression of GPER mRNA indicates a better prognosis for ERα-positive breast cancer; however, its effects in TNBC differ. Whether GPER could serve as a predictive prognostic marker or therapeutic target for TNBC remains unclear. In this review, we provide a detailed introduction to the subcellular localization of GPER, the different effects of various ligands, and the interactions between GPER and closely associated factors in TNBC. We focused on the internal molecular mechanisms specific to TNBC and thoroughly explored the role of GPER in promoting tumor development. We also discussed the interaction of GPER with specific cytokines and chemokines, and the relationship between GPER and immune evasion. Additionally, we discussed the feasibility of using GPER as a therapeutic target in the context of existing studies. This comprehensive review highlights the effects of GPER on TNBC, providing a framework and directions for future research.

PLK4 as a potential target to enhance radiosensitivity in triple-negative breast cancer

Radioresistance is one of the barriers to developing more effective therapies against the most aggressive, triple-negative, breast cancer (TNBC) subtype. In our previous studies, we showed that inhibition of Polo-like Kinase 4 (PLK4) by a novel drug, CFI-400945 significantly enhances the anticancer effects of radiotherapy (RT) compared to single treatment alone. Here we further investigate the role of PLK4 in enhancing radiation effects in TNBC and explore mechanisms of PLK4 inhibition and radiation combinatorial antiproliferative effects. To assess cellular proliferation in response to treatments, we used colony formation assays in TNBC cell lines and patient-derived organoids (PDOs). Downregulation of PLK4 expression was achieved using siRNA silencing in TNBC cell lines. Immunofluorescence against centrin was used to assess the alteration of centriole amplification in response to treatments. We observed that inhibition of PLK4 by CFI-400945 or Centrinone B or its downregulation by siRNA, when combined with RT, resulted in a significant increase in antiproliferative effect in TNBC cells lines and PDOs compared to untreated or single-treated cells. Anticancer synergy was observed using a response matrix in PDOs treated with CFI-400945 and RT. We show that the overamplification of centrioles might be involved in the combined antiproliferative action of RT and PLK4 inhibition. Our data suggest that PLK4 is a promising target for enhancing the anticancer effects of RT in TNBC that, at least in part, is modulated by the overamplification of centrioles. These results support further mechanistic and translational studies of anti-PLK4 agents and RT as an anticancer combination treatment strategy.

Subpopulation treatment effect pattern plot analysis: a prognostic model for distant recurrence-free survival to estimate delayed adjuvant chemotherapy initiation effect in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a heterogeneous disease associated with a poor prognosis. Delaying in time to start adjuvant chemotherapy (TTC) has been related to an increased risk of distant recurrence-free survival (DRFS). We aimed to develop a prognostic model to estimate the effects of delayed TTC among TNBC risk subgroups. We analyzed 687 TNBC patients who received adjuvant chemotherapy at the Instituto Nacional de Enfermedades Neoplasicas (Lima, Peru). Database was randomly divided to create a discovery set (n=344) and a validation set (n=343). Univariate and multivariate Cox regression models were performed to identify prognostic factors for DRFS. Risk stratification was implemented through two models developed based on proportional hazard ratios from significant clinicopathological characteristics. Subpopulation treatment effect pattern plot (STEPP) analysis was performed to determine the best prognostic cut-off points for stratifying TNBC subgroups according to risk scores and estimate Kaplan-Meier differences in 10-year DRFS comparing TTC (≤30 vs.>30 days). In univariate analysis, patients aged ≥70 years (HR=4.65; 95% CI: 2.32-9.34; p=<0.001), those at stages pT3-T4 (HR=3.28; 95% CI: 1.57-6.83; p=0.002), and pN2-N3 (HR=3.00; 95% CI: 1.90-4.76; p=<0.001) were notably associated with higher risk. STEPP analysis defined three risk subgroups for each model. Model N°01 categorized patients into low (score: 0-31), intermediate (score:32-64), and high-risk (score: 65-100) cohorts; meanwhile, Model N°02: low (score: 0-26), intermediate (score: 27-55), and high (score: 56-100). Kaplan-Meier plots showed that in the discovery set, patients with TTC>30 days experienced a 17.5% decrease in 10-year DRFS rate (95%CI=6.7-28.3), and the impact was more remarkable in patients who belong to the high-risk subgroup (53.3% decrease in 10 years-DRFS rate). Similar results were found in the validation set. We developed two prognostic models based on age, pT, and pN to select the best one to classify TNBC. For Model N°02, delayed adjuvant chemotherapy conferred a higher risk of relapse in patients ≥70 years and who were characterized by pT3/T4 and pN2/N3. Thus, more efforts should be considered to avoid delayed TTC in TNBC patients, especially those in high-risk subgroups.

Ginger exosome-like nanoparticles (GELNs) induced apoptosis, cell cycle arrest, and anti-metastatic effects in triple-negative breast cancer MDA-MB-231 cells

Ginger exosome-like nanoparticles (GELNs) have been extensively implicated in alleviating inflammation, maintaining intestinal microbiome and are considered competent drug delivery vehicles. Despite this, the current knowledge of the GELN interaction with cancer cells is limited. Triple-negative breast cancer (TNBC), an aggressive variant lacking efficient therapeutics, necessitates novel natural counterparts with minimal side effects. This study investigates the action of GELNs isolated from ginger rhizomes against TNBC cells. GELNs were isolated by ultracentrifugation and characterized physicochemically. The interaction of GELNs with TNBC cells (MDA-MB-231) was studied in detail. The GELNs induced a concentration-dependent decrease in cell viability in MDA-MB-231 cells without affecting the normal cell lines tested. GELNs induced apoptosis as indicated by morphological changes, nuclear fragmentation, membrane damage, phosphatidyl serine translocation, ROS generation, drop in mitochondrial membrane potential, expression of apoptotic specific proteins, and increased caspase activity. GELNs also instigated cell cycle arrest, retarded cell migration and colony formation in TNBC cells. These findings report a novel action of GELNs against TNBC cells and a closer look at the underlying molecular mechanism of this interspecies communication. This opens newer prospects for using dietary ELNs to target therapeutically challenging cancers.

USP53 Exerts Tumor-Promoting Effects in Triple-Negative Breast Cancer by Deubiquitinating CRKL.

Breast cancer (BC) ranks in the top five malignant tumors in terms of morbidity and mortality rates. Among BC subtypes, TNBC has a high recurrence rate and metastasis rate and the worst prognosis. However, the exact mechanism by which TNBC develops is unclear. Here, we show that the deubiquitinase USP53 contributes to tumor growth and metastasis in TNBC. USP53 is overexpressed in TNBC, and this phenotype is linked to a poor prognosis. Functionally, USP53 promotes TNBC cell proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). More importantly, USP53 decreases the chemosensitivity of BC cells by enhancing v-crk sarcoma virus CT10 oncogene homologue (avian)-like (CRKL) expression. Mechanistically, USP53 directly binds CRKL to stabilize and deubiquitinate it, thereby preventing CRKL degradation. Overall, we discovered that USP53 deubiquitinates CRKL, encourages tumor development and metastasis, and reduces chemosensitivity in TNBC. These findings imply that USP53 might represent a new therapeutic target for the treatment of TNBC.

Proteomics Analysis of Polyphyllin D-Treated Triple-Negative Breast Cancer Cells Reveal the Anticancer Mechanisms of Polyphyllin D

Polyphyllin D (PD), one of the important steroid saponins in traditional medicinal herb Paris polyphylla, has been demonstrated to have anticancer activity both in vitro and in vivo. However, the mechanisms through which PD exerts its anticancer effects in triple-negative breast cancer (TNBC) remain unclear. Our study was presented to evaluate the anticancer effect and the potential mechanisms of PD in two TNBC cell lines, BT-549 and MDA-MB-231. Through comprehensively comparing the liquid chromatography-tandem mass spectrometry (LC–MS/MS) data of PD-treated and untreated BT-549 and MDA-MB-231 cells, we found that PD could induce apoptosis of TNBC cells by activating oxidative phosphorylation pathway in BT-549 cells, as well as inhibiting spliceosome function alteration in MDA-MB-231 cells. These results suggested that the mechanisms underlying the pro-apoptotic effect of PD on TNBC may be cell type-specificity-dependent. Moreover, we found that nodal modulator 2/3 (NOMO2/3) were downregulated both in PD-treated BT-549 and MDA-MB-231 cells, suggesting that NOMO2/3 may be the potential target of PD. Verification experiments revealed that PD deceased NOMO2/3 expression at protein level, rather than mRNA level. Whether NOMO2/3 are the upstream modulators of oxidative phosphorylation pathway and spliceosome needs further validation. In conclusion, a comprehensive proteomics study was performed on PD-treated or untreated TNBC cells, revealing the anticancer mechanisms of PD.

N76-1, a novel CDK7 inhibitor, exhibits potent anti-cancer effects in triple negative breast cancer

Emerging evidence suggests that genetically highly specific triple-negative breast cancer (TNBC) possesses a relatively uniform transcriptional program that is abnormally dependent on cyclin-dependent kinase 7 (CDK7). In this study, we obtained an inhibitor of CDK7, N76-1, by attaching the side chain of the covalent CDK7 inhibitor THZ1 to the core of the anaplastic lymphoma kinase inhibitor ceritinib. This study aimed to elucidate the role and underlying mechanism of N76-1 in TNBC and evaluate its potential value as an anti-TNBC drug. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and colony formation assays showed that N76-1 inhibited the viability of TNBC cells. Kinase activity and cellular thermal shift assays showed that N76-1 directly targeted CDK7. Flow cytometry results revealed that N76-1 induced apoptosis and cell cycle arrest in the G2/M phase. N76-1 also effectively inhibited the migration of TNBC cells by high-content detection. The RNA-seq analysis showed that the transcription of genes, especially those related to transcriptional regulation and cell cycle, was suppressed after N76-1 treatment. Moreover, N76-1 markedly inhibited the growth of TNBC xenografts and phosphorylation of RNAPII in tumor tissues. In summary, N76-1 exerts potent anticancer effects in TNBC by inhibiting CDK7 and provides a new strategy and research basis for the development of new drugs for TNBC.

Dual-targeted and dual-sensitive self-assembled protein nanocarrier delivering hVEGI-192 for triple-negative breast cancer

Breast cancer is a highly prevalent malignancy worldwide among women with an increasing incidence in recent years. Triple-negative breast cancer (TNBC), a specific type of breast cancer, occurs primarily in young women and exhibits large tumor size, high clinical stage, and extremely poor prognosis with a high rate of lymph node, liver, and lung metastases. TNBC is insensitive to endocrine therapy and trastuzumab treatment, and there is an urgent need for effective therapeutics and treatment guidelines. However, investigations into antiangiogenic agents for the treatment of TNBC are ongoing. In this study, we successfully engineered a self-assembled protein nanocarrier TfRBP9-hVEGI-192-ELP fusion protein (TVEFP) to deliver the therapeutic protein, human vascular endothelial growth inhibitor (hVEGI-192). This was found to be effective in inhibiting tumor angiogenesis in vivo. The protein nanocarrier effectively inhibited the progression of TNBC in vivo and showed the behavior of self-assembly, thermoresponsiveness, enzyme stimulation-responsiveness, tumor-targeting, biocompatibility, and biodegradability. Near-infrared imaging studies showed that fluorescent dye-stained TVEFP effectively aggregated at the tumor site. The TVEFP nanocarrier significantly expands the application of the therapeutic protein hVEGI-192 and improves the imaging and biotherapeutic effects in TNBC, chiefly based on anti-angiogenesis effects.

Photosensitive small extracellular vesicles regulate the immune microenvironment of triple negative breast cancer

Currently, the treatment of triple-negative breast cancer (TNBC) is limited by the special pathological characteristics of this disease. In recent years, photodynamic therapy (PDT) has created new hope for the treatment of TNBC. Moreover, PDT can induce immunogenic cell death (ICD) and improve tumor immunogenicity. However, even though PDT can improve the immunogenicity of TNBC, the inhibitory immune microenvironment of TNBC still weakens the antitumor immune response. Therefore, we used the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by TNBC cells to improve the tumor immune microenvironment and enhance antitumor immunity. In addition, bone mesenchymal stem cell (BMSC)-derived sEVs have good biological safety and a strong drug loading capacity, which can effectively improve the efficiency of drug delivery. In this study, we first obtained primary BMSCs and sEVs, and then the photosensitizers Ce6 and GW4869 were loaded into the sEVs by electroporation to produce immunomodulatory photosensitive nanovesicles (Ce6-GW4869/sEVs). When administered to TNBC cells or orthotopic TNBC models, these photosensitive sEVs could specifically target TNBC and improve the tumor immune microenvironment. Moreover, PDT combined with GW4869-based therapy showed a potent synergistic antitumor effect mediated by direct killing of TNBC and activation of antitumor immunity. Here, we designed photosensitive sEVs that could target TNBC and regulate the tumor immune microenvironment, providing a potential approach for improving the effectiveness of TNBC treatment. Statement of SignificanceWe designed an immunomodulatory photosensitive nanovesicle (Ce6-GW4869/sEVs) with the photosensitizer Ce6 to achieve photodynamic therapy and the neutral sphingomyelinase inhibitor GW4869 to inhibit the secretion of small extracellular vesicles (sEVs) by triple-negative breast cancer (TNBC) cells to improve the tumor immune microenvironment and enhance antitumor immunity. In this study, the immunomodulatory photosensitive nanovesicle could target TNBC cells and regulate the tumor immune microenvironment, thus providing a potential approach for improving the treatment effect in TNBC. We found that the reduction in tumor sEVs secretion induced by GW4869 improved the tumor-suppressive immune microenvironment.Moreover, similar therapeutic strategies can also be applied in other kinds of tumors, especially immunosuppressive tumors, which is of great value for the clinical translation of tumor immunotherapy.

Tumor suppressor miR-218 directly targets epidermal growth factor receptor (EGFR) expression in triple-negative breast cancer, sensitizing cells to irradiation

PurposeMicroRNA-218 (miR-218) is a key regulator of numerous processes relevant to tumor progression. In the present study, we aimed to characterize the relationship between miR-218 and the Epidermal Growth Factor Receptor (EGFR) as well as to understand downstream effects in triple-negative breast cancer (TNBC).MethodsWe assessed miR-218 and EGFR expression in cell lines and publicly available primary breast cancer gene expression data. We then overexpressed miR-218 in two TNBC cell lines and investigated effects on EGFR and downstream mitogen-activated protein (MAP) kinase signaling. Luciferase reporter assay was used to characterize a direct binding interaction between miR-218 and EGFR mRNA. Digital holographic microscopy helped investigate cell migration and dry mass after miR-218 overexpression. Cell division and invasion were assessed microscopically, while radiation response after miR-218 overexpression alone or combined with additional EGFR knockdown was investigated via clonogenic assays.ResultsWe found an inverse correlation between EGFR expression and miR-218 levels in cell lines and primary breast cancer tissues. MiR-218 overexpression resulted in a downregulation of EGFR via direct binding of the mRNA. Activation of EGFR and downstream p44/42 MAPK signaling were reduced after pre-miR-218 transfection. Cell proliferation, motility and invasiveness were inhibited whereas cell death and mitotic catastrophe were upregulated in miR-218 overexpressing cells compared to controls. MiR-218 overexpressing and EGFR siRNA-treated cells were sensitized to irradiation, more than miR-218 overexpressing cells alone.ConclusionThis study characterizes the antagonistic relationship between miR-218 and EGFR. It also demonstrates downstream functional effects of miR-218 overexpression, leading to anti-tumorigenic cellular changes.

Abstract 4037: Doxazosin exerts anti-metastatic potential in triple-negative breast cancer via impairment of cancer stem-like features

Abstract Background and Purpose: Triple-negative breast cancer (TNBC) tumors typically harbor a high cancer stem-like population leading to chemo-resistance, recurrence, and metastasis. Tumor metastasis is associated with 90% of cancer-related deaths, highlighting the urgent clinical unmet need. Doxazosin is known to inhibit cell migration and invasion in several cancer cell types; however, the precise mechanisms underlying doxazosin’s anticancer effects in TNBC have not been fully elucidated. In the present study, we sought to investigate the mechanism of action of doxazosin responsible for its effects on apoptosis, cancer stem cell (CSC)-like properties, cell migration, and metastasis in TNBC. Experimental designs: Doxazosin on TNBC cell lines [MDA-MB-231, BT549, and 4T1] in vitro was evaluated in cell viability, apoptosis, cell migration, CD44/CD24 staining, ALDH1 activity, and mammosphere formation. The effect of doxazosin on tumor growth, angiogenesis, and metastasis was evaluated in an orthotopic allograft mice model with CSC-enriched population. Results: Doxazosin significantly reduced cell viability and induced apoptosis in MDA-MB-231 and BT549 cells via activation of caspase-3/-7 and cleavage of PARP. Doxazosin significantly suppressed cell migratory capability, concomitant with disrupting cytoskeletal proteins, including vimentin and F-actin expression in TNBC cells. An impairment of BCSC-like properties was associated with reduction of ALDH1 activity and the CD44+/CD24- population, concomitant with suppression of mammosphere-forming ability. Doxazosin administration reduced tumor growth and lung metastasis, as evidenced by a sharp decline in bioluminescence signal intensity. Inhibitory effect of tumor growth was accompanied by a significant decrease of Ki-67 and enhancement of apoptosis with DNA fragmentation and increased cleaved-caspase-3 expression. The latter phenomenon was associated with the impediment of JAK2/STAT3 signaling pathway and CSC-like properties. Furthermore, no toxic effects of doxazosin were found in liver and kidney function in animals. Conclusion: Taken together, our findings highlight doxazosin as a promising candidate for drug repurposing in suppressing metastatic TNBC. Citation Format: Seongjae Kim, Dongmi Ko, Juyeon Seo, Soeun Park, Minsu Park, Kee Dal Nam, Yong koo Kang, So Ra Seock, Jaeyoun Park, Eunhye Oh, Eunsun Jung, Yoon-Jae Kim, Ji Young Kim, Jae Hong Seo. Doxazosin exerts anti-metastatic potential in triple-negative breast cancer via impairment of cancer stem-like features. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4037.

Abstract 6654: Association PD-L1 overexpression with immune checkpoint inhibitor effect in triple-negative breast cancer

Abstract Background: Recently, studies on the antitumor effects of PD-L1 inhibitors have gained importance in difficult to treat solid tumors. In particular, the expression of PD-L1 could be an important factor in evaluating the possibility of immunotherapy in triple-negative breast cancer where targeted therapy is not possible and the prognosis is poor. Accordingly, this study investigated the effect of PD-L1 overexpression on the immune checkpoint inhibitor effect in triple-negative breast cancer. Method: The relationship between T stage and N stage according to PD-L1 mRNA expression was analyzed in the TCGA and METABRIC datasets of breast cancer. To evaluate the role of PD-L1, PD-L1-overexpressing breast cancer cell lines for in vitro and in vivo experiments were established. Result: Using TCGA and METABRIC datasets, we found that PD-L1 showed the highest expression in breast cancer of the basal subtype, but the correlation between T stage and N stage according to PD-L1 expression was not statistically significant. To determine the functional importance of PD-L1 in breast cancer, we established 4T1-PD-L1-overexpressing breast cancer cell lines. We observed that the overexpression of PD-L1 promoted cell proliferation, migration, and invasion. In the orthotopic tumor mouse model, PD-L1 showed no significant difference in tumor growth compared to the control group, but anti-PD-L1 treatment showed a significant antitumor effect. Conclusion: We confirmed that overexpression of PD-L1 in an orthotopic breast cancer model did not induce increased tumor growth. Above all, it was confirmed that a high PD-L1 level enhances the antitumor effect of immune checkpoint inhibitors. Our findings show that the expression level of PD-L1 in breast cancer has a significant effect on the immune checkpoint inhibitor effect. We need to further investigate the role of PD-L1 in breast cancer. Citation Format: A Young Park, Ju Hee Kim, Sangen Lee, Hong Kyu Kim, Han-Byoel Lee, Wonshick Han. Association PD-L1 overexpression with immune checkpoint inhibitor effect in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6654.

Nanosecond Pulsed Electric Field Induces an Antitumor Effect in Triple-Negative Breast Cancer via CXCL9 Axis Dependence in Mice

Triple-negative breast cancer (TNBC) is a refractory tumor, and therapeutic options are very limited. Local ablation has been applied recently. Chemokines play a critical role in the recruitment of immune cells into ablative tumors. Nanosecond pulsed electric field (nsPEF) shows potential anti-tumor efficacy, but the mechanism for maintaining the immune effect is not very clear. Here, we applied nsPEF for treating 4T1 breast cancer cells in vitro. RNA sequencing (RNA-seq) was applied. Anti-CXCL9 was used alone or combined with nsPEF to treat triple-negative breast cancer in mice. We demonstrated that nsPEF effectively induced cell apoptosis and inhibited the growth and metastasis of triple-negative breast cancer. An immune effect, especially chemotaxis, was activated by nsPEF. The number of infiltrated CD8+ T cells was increased significantly. We found that the inhibition of residual breast cancer growth by nsPEF was dependent on the CXCL9 axis. In conclusion, our work demonstrated that nsPEF effectively ablated the tumor, aroused an immune response, and inhibited residual breast cancer growth via CXCL9 axis dependence in mice.