Triple Negative Cell Lines Research Articles

Discovery of a potent and selective PARP1 degrader promoting cell cycle arrest via intercepting CDC25C-CDK1 axis for treating triple-negative breast cancer

PARP1 is a multifaceted component of DNA repair and chromatin remodeling, making it an effective therapeutic target for cancer therapy. The recently reported proteolytic targeting chimera (PROTAC) could effectively degrade PARP1 through the ubiquitin–proteasome pathway, expanding the therapeutic application of PARP1 blocking. In this study, a series of nitrogen heterocyclic PROTACs were designed and synthesized through ternary complex simulation analysis based on our previous work. Our efforts have resulted in a potent PARP1 degrader D6 (DC50 = 25.23 nM) with high selectivity due to nitrogen heterocyclic linker generating multiple interactions with the PARP1-CRBN PPI surface, specifically. Moreover, D6 exhibited strong cytotoxicity to triple negative breast cancer cell line MDA-MB-231 (IC50 = 1.04 µM). And the proteomic results showed that the antitumor mechanism of D6 was found that intensifies DNA damage by intercepting the CDC25C-CDK1 axis to halt cell cycle transition in triple-negative breast cancer cells. Furthermore, in vivo study, D6 showed a promising PK property with moderate oral absorption activity. And D6 could effectively inhibit tumor growth (TGI rate = 71.4 % at 40 mg/kg) without other signs of toxicity in MDA-MB-321 tumor-bearing mice. In summary, we have identified an original scaffold and potent PARP1 PROTAC that provided a novel intervention strategy for the treatment of triple-negative breast cancer.

Combination of verteporfin-photodynamic therapy with 5-aza-2'-deoxycytidine enhances the anti-tumour immune response in triple negative breast cancer.

Triple negative breast cancer (TNBC) is a subtype of breast cancer characterised by its high tumourigenic, invasive, and immunosuppressive nature. Photodynamic therapy (PDT) is a focal therapy that uses light to activate a photosensitizing agent and induce a cytotoxic effect. 5-aza-2'-deoxycytidine (5-ADC) is a clinically approved immunomodulatory chemotherapy agent. The mechanism of the combination therapy using PDT and 5-ADC in evoking an anti-tumour response is not fully understood. The present study examined whether a single dose of 5-ADC enhances the cytotoxic and anti-tumour immune effect of low dose PDT with verteporfin as the photosensitiser in a TNBC orthotopic syngeneic murine model, using the triple negative murine mammary tumour cell line 4T1. Histopathology analysis, digital pathology and immunohistochemistry of treated tumours and distant sites were assessed. Flow cytometry of splenic and breast tissue was used to identify T cell populations. Bioinformatics were used to identify tumour immune microenvironments related to TNBC patients. Functional experiments showed that PDT was most effective when used in combination with 5-ADC to optimize its efficacy. 5-ADC/PDT combination therapy elicited a synergistic effect in vitro and was significantly more cytotoxic than monotherapies on 4T1 tumour cells. For tumour therapy, all types of treatments demonstrated histopathologically defined margins of necrosis, increased T cell expression in the spleen with absence of metastases or distant tissue destruction. Flow cytometry and digital pathology results showed significant increases in CD8 expressing cells with all treatments, whereas only the 5-ADC/PDT combination therapy showed increase in CD4 expression. Bioinformatics analysis of in silico publicly available TNBC data identified BCL3 and BCL2 as well as the following anti-tumour immune response biomarkers as significantly altered in TNBC compared to other breast cancer subtypes: GZMA, PRF1, CXCL1, CCL2, CCL4, and CCL5. Interestingly, molecular biomarker assays showed increase in anti-tumour response genes after treatment. The results showed concomitant increase in BCL3, with decrease in BCL2 expression in TNBC treatment. In addition, the treatments showed decrease in PRF1, CCL2, CCL4, and CCL5 genes with 5-ADC and 5-ADC/PDT treatment in both spleen and breast tissue, with the latter showing the most decrease. To our knowledge, this is the first study that shows which of the innate and adaptive immune biomarkers are activated during PDT related treatment of the TNBC 4T1 mouse models. The results also indicate that some of the immune response biomarkers can be used to monitor the effectiveness of PDT treatment in TNBC murine model warranting further investigation in human subjects.

New Organotin (IV) Compounds Derived from Dehydroacetic Acid and Thiosemicarbazides: Synthesis, Rational Design, Cytotoxic Evaluation, and Molecular Docking Simulation.

Organotin complexes were prepared through a one-pot reaction with three components by reacting thiosemicarbazide or 4-methyl-3-thiosemicarbazide or 4-phenylthiosemicarbazide, dehydroacetic acid (DHA) and dibutyl, diphenyl, dicyclohexyl, and bis[(trimethylsilyl)methyl]tin(IV) oxides; all complexes were characterized by infrared (IR), ultraviolet-visible (UV-vis), mass spectrometry (MS), and nuclear magnetic resonance (NMR) spectroscopy. The 119Sn NMR revealed chemical shifts corresponding to a pentacoordinated environment in solution. The X-ray crystallography of the two complexes evidenced the formation of monomeric complexes with a pentacoordinated geometry around tin via three donor atoms from the ligand, the sulfur of the thiol, the nitrogen of the imine group, and the oxygen of the pyran ring. The geometries of the five-coordinated complexes 3a (Bu2SnL3), 3c (Ph2SnL3), and 3d (Cy2SnL3) acid were intermediate between square pyramidal and trigonal bipyramidal, and complex 1a (Bu2SnL1) adopted a bipyramidal trigonal geometry (BPT). The sulforhodamine B assay assessed the cytotoxicity of organotin(IV) complexes against the MDA-MB-231 and MCF-7 (human mammary adenocarcinoma) cell lines and one normal COS-7 (African green monkey kidney fibroblast). The IC50 values evidenced a significant antiproliferative effect on cancer cells; the complexes were more potent than the positive cisplatin control and the corresponding ligands, dehydroacetic acid thiosemicarbazone (L1), dehydroacetic acid-N(4)-methylthiosemicarbazone (L2), and dehydroacetic acid-N(4)-phenylthiosemicarbazone (L3). The IC50 values also indicated that the organotin(IV) complexes were more cytotoxic against the triple-negative breast cell line MDA-MB-231 than MCF-7, inducing significant morphological alterations. The interactions of organotin(IV) 1c (Ph2SnL1), 1d (Cy2SnL1), and 1e (((CH3)3SiCH2)2SnL1) were evaluated with ss-DNA by fluorescence; intensity changes of the fluorescence were indicative of the displacement of ethidium bromide (EB), confirming the interaction of the organotin(IV) complexes with ss-DNA; the results showed a DNA binding affinity. The thermodynamic parameters obtained through isothermal titration calorimetry showed that the interaction of 1c (Ph2SnL1), with ss-ADN, was exothermic. Molecular docking studies also demonstrated that the organotin(IV) complexes were intercalated in DNA by conventional hydrogen bonds, carbon-hydrogen bonds, and π-alkyl interactions. These complexes furthermore showed a greater affinity towards DNA than cisplatin.

PRIMA-1 synergizes olaparib-induced cell death in p53 mutant triple negative human breast cancer cell line via restoring p53 function, arresting cell cycle, and inducing apoptosis.

This study concerned with assessing the effect of restoring p53 using PRIMA-1 on the anti-cancer activity of olaparib against TP53-mutant triple negative breast cancer (TNBC) cells and exploring the optimum synergistic concentrations and the underlying mechanism. Human BC cell lines, MDA-MB-231 with mutated TP53 gene, and MCF-7 with wild-type TP53 gene were treated with olaparib and/or PRIMA-1. The IC50 value for olaparib was significantly decreased by PRIMA-1 in MDA-MB-231 cells compared to MCF-7 cells. Contrary to MCF-7 cells, co-treatment with olaparib and PRIMA-1 had a synergistic anti-proliferative effect in MDA-MB-231 at all tested concentrations with the best synergistic combination at 45and 8.5µM, respectively, and furthermore PRIMA-1 enhanced olaparib-induced apoptosis. This synergistic apoptotic effect was associated with a significant boost in mRNA expression of TP53 gene, cell cycle arrest at G2/M phase, modulation of BRCA-1, BAX and Bcl2 proteins expressions, and induction of active caspase-3. These results present a clue for the utility of combined olaparib and PRIMA-1 in treatment of TP53-mutant TNBC invitro. PRIMA-1 triggers olaparib-induced MDA-MB-231 cell death in a synergistic manner via restoring TP53, decreasing BRCA-1 expression, cell cycle arrest, and enhancement of apoptosis via p53/BAX/Bcl2/caspase 3 pathway.

THU483 Role Of Excess GH On Breast Cancer Aggressiveness And Metastasis In Acromegaly

Abstract Disclosure: J. Oh: None. C. Kang: None. E. Wang: None. S. Lee: None. J. Nam: None. C. Ku: None. E. Lee: None. Introduction Acromegaly is a rare disease characterized by chronic growth hormone (GH) and insulin-like growth factor-1 hypersecretion. In previous studies, it is known that the incidence of breast cancer increases in patients with acromegaly. For breast cancers, autocrine GH and GHR are expressed in primary lesion, which is significantly increased in metastatic one. To date, however, the precise mechanism of endocrine GH in breast cancer metastasis has not been defined. This study aimed to investigate the role of endocrine excess GH facilitating breast cancer metastasis in acromegaly mice model. Method We used Aiplox/lox (Con) and rGHp-Cretg/+; Aiplox/lox mice (Acro), which was previously established to model the phenotype of human acromegaly. To generate orthotopic xenograft model, we used C57BL/6 female mouse derived breast cancer cell line Py230 cells. Py230 cells were injected into mammary gland and tumor volumes were measured every 2 days. Tumor growth and metastasis were confirmed by bioluminescent in vivo imaging system (IVIS). Also, we investigated the effects of GH on triple negative breast cancer cell lines, which have the higher expression of GHR. To define GH induced pathway, we performed RT2 array. Cell proliferation was investigated with MTT assay and cell migration was investigated with wound healing assay and transwell invasion assay. Results In mouse orthotopic xenograft studies, tumor volumes of Acro mice were higher than that of Con mice (increase of 310%, 846.04±290.7 vs. 205.97±33.7 mm3, p=0.001). The tumor volume and serum GH level showed positive correlation (R2= 0.4739, p=0.027). After 14 weeks, the Acro mice showed significantly increased bioluminescence activity in the lung (increase of 1023%, 494666.7±104668.2 vs. 44037.7±6641.9 AU, p=0.0127). When GHR antagonist was treated, tumor growth and lung metastasis were decreased. We excised primary tumor and lung metastatic foci and performed RT2 array. We found Tcf-20 gene, which is upregulated in both primary tumor and lung metastatic burden (increase of 216%, p=0.0153). We confirmed GH induced Tcf-20 gene induction (increase of 57% (p=0.043), 41% (p=0.05), 67% (p=0.0021) in Hs578T, Py230 (p=0.05), and Hcc1806 cells each). We knocked down Ghr or Tcf-20 genes and observed decrease of invasion and migration compared to GH treated siCON HCC1806 (decrease of 64.5% in siGHR, decrease of 83.3% in siTCF-20, compared to siCON cells, p=0.000819977) and Py230 cells (decrease of 47.3% in siGHR transfected, decrese of 49.3% in siTCF-20 transfected, compared to siCON cells, p=0.0012). Conclusion Excess GH in acromegaly possess a strong potential to promotes tumor development and metastasis. We identified a molecular mechanism by which GH-induced TCF-20 promote tumor aggression and metastasis. These findings can aid in the development of improved strategies for managing cancer in acromegalic patients. Presentation: Thursday, June 15, 2023

Design of PLGA nanoparticles for sustained release of hydroxyl-FK866 by microfluidics

The use of nanoparticle (NP) delivery systems in cancer treatment has received significant interest, however use of such systems in delivery of cytotoxic chemotherapy agents can be limited by low encapsulation efficiency and burst release of the cytotoxin, as well issues with throughput and reproducibility during the fabrication of drug-loaded NPs. In this study, we used a hydrodynamic flow-focusing microfluidic system to successfully produce poly(lactic-co-glycolic acid) (PLGA) NPs. The physico-chemical properties of PLGA NPs were controlled by changing the manufacturing parameters, such as flow rate ratio, total flow rate, PLGA and surfactant concentration. The NAMPT inhibitor-polymer conjugate, hydroxyl-FK866-PLGA, was synthesized and used to fabricate hydroxyl-FK866-PLGA NPs for the formulation of localized delivery systems able to release low doses of cytotoxins and enhance the efficacy of NAMPT inhibitors. Hydroxyl-FK866-PLGA NPs were prepared with optimized fabrication parameters, having average Z-size of 128 ± 8 nm (PDI < 0.2), ζ-potential of −14.8 ± 5.3 mV and high encapsulation efficiency (98.6 ± 5.8 %). The pH-dependent release of hydroxyl-FK866 was monitored over time in conditions mimicking the normal (pH 7.4) and inflamed/tumor (pH 6.4) microenvironments, observing a sustained release pattern (over two months) without any initial burst release. Finally, toxicity of hydroxyl-FK866-PLGA NPs were tested in selected human cell lines, the human leukemia monocytic cell line (THP-1), and the human triple negative breast cancer cell line (MDA-MB-231). Our work suggests that microfluidic systems are a promising technology for a rapid and efficient manufacturing of PLGA-based NPs for the controlled release of cytotoxins. Moreover, the use of drug-polymer conjugates is an effective approach for the manufacturing of polymeric NPs enabling high encapsulation efficiency and a prolonged and sustained pH-dependent drug release.

Histone methyltransferase inhibitor UNC0642 promotes breast cancer cell death by upregulating TXNIP-dependent oxidative stress

Breast cancer (BC) is one of the most frequent type of cancer in women worldwide. Current therapeutic strategies for BC are not always effective. In this study, we investigated the anticancer activity of an epigenetic compound UNC0642 and its mechanism of action in suppressing BC cell growth and survival. UNC0642 was developed as a selective inhibitor of G9a that is responsible for histone H3K9 methylation. After screening different BC cell lines, we found UNC0642 had the lowest IC-50 against MDA-MB-231 cells, a triple-negative BC cell line. To identify additional UNC0642 targets, we performed RNA-seq analyses in BC cells following UNC0642 treatment. UNC0642 significantly upregulated mRNA expression of thioredoxin-interacting protein (TXNIP), which was also validated by western blotting. We further showed that TXNIP upregulation was associated with dose-dependent elevation of reactive oxygen species, concurrent with loss of mitochondrial membrane potential and activation of caspase-3-dependent apoptosis. Finally, we demonstrated that UNC0642 treatment induced BC cell apoptosis in vitro and suppression of tumor growth in xenograft mouse models that was coupled with TXNIP activation. Taken together, our results show that UNC0642 exerts its antitumor function via upregulating TXNIP expression and oxidative stress to impair mitochondrial function and induce caspase-dependent cell death. This observation could inform future breast cancer therapies by targeting TXNIP-dependent ROS signaling.

Synthesis of Novel Pyrazole-Oxindole Conjugates with Cytotoxicity in Human Cancer Cells via Apoptosis.

A novel series of pyrazole-oxindole conjugates were prepared and characterized as potential cytotoxic agents by FT-IR, NMR and HR-MS. The cytotoxic activity of these compounds was tested in the Jurkat acute T cell leukemia, CEM acute lymphoblastic leukemia, MCF10 A mammary epithelial and MDA-MB 231 triple negative breast cancer cell lines. Among the tested conjugates, 5-methyl-3-((3-(1-phenyl)-3-(p-tolyl)-1H-pyrazol-4-yl)methylene)indolin-2-one 6h emerged as the most cytotoxic with a CC50 of 4.36+/-0.2 μM against Jurkat cells. The mechanism of cell death induced by 6h was investigated through the Annexin V-FITC assay via flow cytometry. Reactive oxygen species (ROS) accumulation, mitochondrial health and the cell cycle progression were also evaluated in cells exposed to 6h. Results demonstrated that 6h induces apoptosis in a dose-response manner, without generating ROS and/or altering mitochondrial health. In addition, 6h disrupted the cell cycle distribution causing an increase in DNA fragmentation (Sub G0-G1), and an arrest in the G0-G1 phase. Taken together, the 6h compound revealed a strong potential as an antineoplastic agent evidenced by its cytotoxicity in leukemia cells, the activation of apoptosis and restriction of the cell cycle progression.

PRIMA-1MET Does Not Restore Vitamin D Sensitivity to MDA-MB-231 and MDA-MB-468 Triple-Negative Breast Cancer Cells.

Vitamin D is a steroid hormone that causes growth suppression in cultured cells. We had previously discovered that the triple-negative breast cancer cell lines MDA-MB-231 and MDA-MB-468 did not have growth suppression with vitamin D, while MCF-7 did. MCF-7 cells are not triple-negative and have wild-type p53. Both MDA-MB-231 and MDA-MB-468 have mutations in p53 and these mutations were a possible explanation for the lack of growth suppression with vitamin D. Our hypothesis was that reactivation of p53 in the triple-negative cell lines would cause them to become sensitive to vitamin D. We chose to use the small molecule PRIMA-1MET to reactivate p53 as it has been previously shown to restore function to the p53 mutants present in MB-231 and MB-468. We then measured the ability of vitamin D and its analogues calcipotriol and EB1089 to suppress growth in the presence of PRIMA-1MET. Here, we show that while PRIMA-1MET can kill the breast cancer cells investigated in this study, it does not restore their sensitivity to vitamin D or its analogues.

In-vitro anticancer evaluation of newly designed and characterized tri/tetra-substituted imidazole congeners- maternal embryonic leucine zipper kinase inhibitors: Molecular docking and MD simulation approaches

Our cascading attempt to develop new potent molecules now involves designing a series of imidazole derivatives and synthesizing two sets of 2,4,5- tri-substituted (4a–4d) and 1,2,4,5-tetra-substituted (6a–6d) imidazole by the principle of Debus-Radziszewski multicomponent synthesis reaction. The structures of the obtained compounds were confirmed by 1H/13C NMR, FT-IR, elemental analysis, purity and the retention time was analyzed by HPLC. Based upon the binding affinity in the molecular docking studies, we have synthesized different imidazole derivatives from which compound 6c have been found to show more anti-proliferative activity by inducing apoptosis at a higher rate than the other compounds corroborating the in-silico prediction. The structure and crystallinity of compound 4d have been confirmed by single XRD analysis. The synthesized molecules were screened for their in vitro anti-cancer properties in triple negative breast cancer cell line (MDA-MB-231), pancreatic cancer cell lines (MIA PaCa-2) and oral squamous cell carcinoma cell line (H357) and results indicated that all the compounds inhibited the cell proliferation in a concentration-dependent manner at different time points. The compounds 4b and 6d were found to be effective against the S. aureus bacterial strain whereas only compound 4d fairly inhibited the fungal strain of T. rubrum with a MIC 12.5 μg/mL. Molecular docking study reveals good interaction of the synthesized compounds with known target MELK involved in oncogenesis having high binding profiles. The lead compound 6c was further analyzed by the detailed molecular dynamics study to establish the stability of the ligand–enzyme complex.

Novel Iron Chelator SK4 Drives Cytotoxicity through Inhibiting Mitochondrial Metabolism in Ovarian and Triple Negative Breast Cancer Cell Lines.

Anti-cancer therapy by iron chelation has been shown to inhibit many cellular processes including DNA replication, mitochondrial metabolism and oncogenic signalling pathways (e.g., EGFR). Iron chelator SK4 represents a double pronged approach towards treating cancer. SK4 enters through LAT1, a commonly overexpressed amino acid transporter in tumours, thus targeting iron addiction and LAT1 overexpression. The aim of this study was to characterise the mode of action of SK4 through proteomics, metabolomics, lipidomics and seahorse real-time analysis in ovarian cell line SKOV3 and triple negative breast cancer cell line MDA MB 231. Pathway enrichment of proteomics data showed an overrepresentation of metabolism related pathways. Metabolic change after SK4 exposure have been confirmed in investigations of changes in basal and maximal mitochondrial respiration using seahorse real-time analysis of mitochondrial metabolism. Metabolomics also showed an increase in AMP and glucose-1-phosphate. Interestingly, our lipidomics data show a decrease in phospholipid synthesis in the SKOV3 cells which is in contrast with previous data which showed an upregulation of ceramide driven apoptosis. In summary, our data highlight impairment of energy metabolism as a mechanism of action underlying SK4 apoptosis, but also suggest a potential role of ceramide induction in the phenotypic outcome of the cell model.

In vivo optical imaging of tumor stromal cells with hypoxia-inducible factor activity.

Tumors contain various stromal cells, such as immune cells, endothelial cells, and fibroblasts, which contribute to the development of a tumor-specific microenvironment characterized by hypoxia and inflammation, and are associated with malignant progression. In this study, we investigated the activity of intratumoral hypoxia-inducible factor (HIF), which functions as a master regulator of the cellular response to hypoxia and inflammation. We constructed the HIF activity-monitoring reporter gene hypoxia-response element-Venus-Akaluc (HVA) that expresses the green fluorescent protein Venus and modified firefly luciferase Akaluc in a HIF activity-dependent manner, and created transgenic mice harboring HVA transgene (HVA-Tg). In HVA-Tg, HIF-active cells can be visualized using AkaBLI, an ultra-sensitive in vivo bioluminescence imaging technology that produces an intense near-infrared light upon reaction of Akaluc with the D-luciferin analog AkaLumine-HCl. By orthotopic transplantation of E0771, a mouse triple negative breast cancer cell line without a reporter gene, into HVA-Tg, we succeeded in noninvasively monitoring bioluminescence signals from HIF-active stromal cells as early as 8 days after transplantation. The HIF-active stromal cells initially clustered locally and then spread throughout the tumors with growth. Immunohistochemistry and flow cytometry analyses revealed that CD11b+ F4/80+ macrophages were the predominant HIF-active stromal cells in E0771 tumors. These results indicate that HVA-Tg is a useful tool for spatiotemporal analysis of HIF-active tumor stromal cells, facilitating investigation of the roles of HIF-active tumor stromal cells in tumor growth and malignant progression.

Impact of AKT1 on cell invasion and radiosensitivity in a triple negative breast cancer cell line developing brain metastasis.

The PI3K/AKT pathway is activated in 43-70% of breast cancer (BC)-patients and promotes the metastatic potential of BC cells by increasing cell proliferation, invasion and radioresistance. Therefore, AKT1-inhibition in combination with radiotherapy might be an effective treatment option for triple-negative breast cancer (TNBC)-patients with brain metastases. The impact of AKT1-knockout (AKT1_KO) and AKT-inhibition using Ipatasertib on MDA-MB-231 BR cells was assessed using in vitro cell proliferation and migration assays. AKT1-knockout in MDA-MB-231BR cells was performed using CRISPR/Cas9. The effect of AKT1-knockout on radiosensitivity of MDA-MB-231BR cell lines was determined via colony formation assays after cell irradiation. To detect genomic variants in AKT1_KO MDA-MB-231BR cells, whole-genome sequencing (WGS) was performed. Pharmacological inhibition of AKT with the pan-AKT inhibitor Ipatasertib led to a significant reduction of cell viability but did not impact cell migration. Moreover, only MDA-MB-231BR cells were sensitized following Ipatasertib-treatment. Furthermore, specific AKT1-knockout in MDA-MB-231BR showed reduced cell viability in comparison to control cells, with significant effect in one of two analyzed clones. Unexpectedly, AKT1 knockout led to increased cell migration and clonogenic potential in both AKT1_KO clones. RNAseq-analysis revealed the deregulation of CTSO, CYBB, GPR68, CEBPA, ID1, ID4, METTL15, PBX1 and PTGFRN leading to the increased cell migration, higher clonogenic survival and decreased radiosensitivity as a consequence of the AKT1 knockout in MDA-MB-231BR. Collectively, our results demonstrate that Ipatasertib leads to radiosensitization and reduced cell proliferation of MDA-MB-231BR. AKT1-inhibition showed altered gene expression profile leading to modified cell migration, clonogenic survival and radioresistance in MDA-MB-231BR. We conclude, that AKT1-inhibition in combination with radiotherapy contribute to novel treatment strategies for breast cancer brain metastases.

Fractions of Methanol Extracts from the Resurrection Plant Haberlea rhodopensis Have Anti-Breast Cancer Effects in Model Cell Systems

Breast cancer is among the most problematic diseases and a leading cause of death in women. The methods of therapy widely used, so far, are often with many side effects, seriously hampering patients’ quality of life. To overcome these constraints, new cancer treatment alternatives are constantly tested, including bioactive compounds of plant origin. Our aim was to study the effects of Haberlea rhodopensis methanol extract fractions on cell viability and proliferation of two model breast cancer cell lines with different characteristics. In addition to the strong reduction in cell viability, two of the fractions showed significant influence on the proliferation rate of the hormone receptor expressing MCF7 and the triple negative MDA-MB231 breast cancer cell lines. No significant effects on the benign MCF10A cell line were observed. We applied a large scale non-targeted approach to purify and identify highly abundant compounds from the active fractions of H. rhodopensis extracts. By the combined NMR/MS approach, myconoside was identified in the fractions and hispidulin 8-C-(6-O-acetyl-2″-O-syringoyl-β-glucopyranoside) was found in one of them. We further performed molecular docking analysis of possible myconoside interactions with several proteins, important for breast cancer proliferation. High probability of binding was established for GLUT1 transporter, estrogen receptor and MYST acetyltransferase. Our results are a good background for future studies on the use of myconoside for targeted breast cancer therapy.

Sertraline as a potential cancer therapeutic approach: Biological relevance of TCTP in breast cancer cell lines and tumors

PurposeThis study aimed to evaluate the role of Translationally Controlled Tumor Protein (TCTP) in breast cancer (BC) and investigate the effects of sertraline, a serotonin selective reuptake inhibitor (SSRI), on BC cells. The objective was to assess the potential of sertraline as a therapeutic agent in BC treatment by examining its ability to inhibit TCTP expression and exert antitumor effects. Material and MethodsWe utilized five different BC cell lines representing the molecular heterogeneity and distinct subtypes of BC, including luminal, normal-like, HER2-positive, and triple-negative BC. These subtypes play a crucial role in determining clinical treatment strategies and prognosis. ResultsThe highest levels of TCTP were observed in triple-negative BC cell lines, known for their aggressive behavior. Sertraline treatment reduced TCTP expression in BC cell lines, significantly impacting cell viability, clonogenicity, and migration. Additionally, sertraline sensitized triple-negative BC cell lines to cytotoxic chemotherapeutic drugs (doxorubicin and cisplatin) suggesting its potential as an adjunctive therapy to enhance the chemotherapeutic response. Bioinformatic analysis of TCTP mRNA levels in TCGA BC data revealed a negative correlation between TCTP levels and patient survival, as well as between TCTP/tpt1 and Ki67. These findings contradict our data and previous studies indicating a correlation between TCTP protein levels and aggressiveness and poor prognosis in BC. ConclusionsSertraline shows a promise as a potential therapeutic option for BC, particularly in triple-negative BC. Its ability to inhibit TCTP expression, enhance chemotherapeutic response, highlights its potential clinical utility in BC treatment, specifically in triple-negative BC subtype.

Optimized Modeling of Metastatic Triple-Negative Invasive Lobular Breast Carcinoma.

Invasive lobular carcinoma (ILC) is a common breast cancer subtype that is often diagnosed at advanced stages and causes significant morbidity. Late-onset secondary tumor recurrence affects up to 30% of ILC patients, posing a therapeutic challenge if resistance to systemic therapy develops. Nonetheless, there is a lack of preclinical models for ILC, and the current models do not accurately reproduce the complete range of the disease. We created clinically relevant metastatic xenografts to address this gap by grafting the triple-negative IPH-926 cell line into mouse milk ducts. The resulting intraductal xenografts accurately recapitulate lobular carcinoma in situ (LCIS), invasive lobular carcinoma, and metastatic ILC in relevant organs. Using a panel of 15 clinical markers, we characterized the intratumoral heterogeneity of primary and metastatic lesions. Interestingly, intraductal IPH-926 xenografts express low but actionable HER2 and are not dependent on supplementation with the ovarian hormone estradiol for their growth. This model provides a valuable tool to test the efficiency of potential new ILC therapeutics, and it may help detect vulnerabilities within ILC that can be exploited for therapeutic targeting.

Computational drug repositioning for the identification of new agents to sensitize drug-resistant breast tumors across treatments and receptor subtypes.

Drug resistance is a major obstacle in cancer treatment and can involve a variety of different factors. Identifying effective therapies for drug resistant tumors is integral for improving patient outcomes. In this study, we applied a computational drug repositioning approach to identify potential agents to sensitize primary drug resistant breast cancers. We extracted drug resistance profiles from the I-SPY 2 TRIAL, a neoadjuvant trial for early stage breast cancer, by comparing gene expression profiles of responder and non-responder patients stratified into treatments within HR/HER2 receptor subtypes, yielding 17 treatment-subtype pairs. We then used a rank-based pattern-matching strategy to identify compounds in the Connectivity Map, a database of cell line derived drug perturbation profiles, that can reverse these signatures in a breast cancer cell line. We hypothesize that reversing these drug resistance signatures will sensitize tumors to treatment and prolong survival. We found that few individual genes are shared among the drug resistance profiles of different agents. At the pathway level, however, we found enrichment of immune pathways in the responders in 8 treatments within the HR+HER2+, HR+HER2-, and HR-HER2- receptor subtypes. We also found enrichment of estrogen response pathways in the non-responders in 10 treatments primarily within the hormone receptor positive subtypes. Although most of our drug predictions are unique to treatment arms and receptor subtypes, our drug repositioning pipeline identified the estrogen receptor antagonist fulvestrant as a compound that can potentially reverse resistance across 13/17 of the treatments and receptor subtypes including HR+ and triple negative. While fulvestrant showed limited efficacy when tested in a panel of 5 paclitaxel resistant breast cancer cell lines, it did increase drug response in combination with paclitaxel in HCC-1937, a triple negative breast cancer cell line. We applied a computational drug repurposing approach to identify potential agents to sensitize drug resistant breast cancers in the I-SPY 2 TRIAL. We identified fulvestrant as a potential drug hit and showed that it increased response in a paclitaxel-resistant triple negative breast cancer cell line, HCC-1937, when treated in combination with paclitaxel.

Therapeutic effects of guanidine hydrochloride on breast cancer through targeting KCNG1 gene

BackgroundTriple-negative breast cancer (TNBC) is one of the subtypes of breast cancer (BC) that is associated with poor survival rates and failure to respond to hormonal and targeted therapies. ObjectiveThe aim of this study was to identify a specific gene at the expression level for TNBC and targeting of this type of breast cancer based on it. Using TCGA database, genes that are particularly high expression in TNBC subtypes compared to other BC subtypes (in terms of receptor status) and normal samples were identified and their sensitivity and specificity were evaluated. Using PharmacoGX and Drug Bank data, drug sensitivity and drug-appropriate genes were identified, respectively. The effects of the identified drug on triple-negative cell lines (MDA-MB-468) were evaluated in comparison with the cell line of other subtypes (MCF7) by apoptosis and MTS tests. ResultsData analyzes showed that the expression level of KCNG1 gene in the TNBC subgroup was significantly higher compared to other BC subtypes from the KCN gene family and ROC results showed that this gene had highest sensitivity and specificity in TNBC subtype. The results of drug resistance and sensitivity showed that an increase in the expression level of KCNG1 was associated with sensitivity to Cisplatin and Oxaliplatin. Moreover, Drug Bank results showed that Guanidine hydrochloride (GuHCl) was a suitable inhibitor for KCNG1. In vitro results showed that the expression level of KCNG1 was higher in MDA-MB-468 compared to MCF7. In addition, the rate of apoptosis in response to GuHCl treatment in MDA-MB-468 cell line as TNBC cell model was higher than MCF7 in the same concentration. ConclusionThis study revealed that GuHCl could be a suitable treatment for TNBC subtype by targeting of KCNG1.

The inhibition of steroid hormones determines the fate of IPC-366 tumor cells, highlighting the crucial role of androgen production in tumor processes

Inflammatory mammary cancer (IMC) is a disease that affects female dogs. It is characterized by poor treatment options and no efficient targets. However, anti-androgenic and anti-estrogenic therapies could be effective because IMC has a great endocrine influence, affecting tumor progression. IPC-366 is a triple negative IMC cell line that has been postulated as a useful model to study this disease. Therefore, the aim of this study was to inhibit steroid hormones production at different points of the steroid pathway in order to determine its effect in cell viability and migration in vitro and tumor growth in vivo. For this purpose, Dutasteride (anti-5αReductase), Anastrozole (anti-aromatase) and ASP9521 (anti-17βHSD) and their combinations have been used. Results revealed that this cell line is positive to estrogen receptor β (ERβ) and androgen receptor (AR) and endocrine therapies reduce cell viability. Our results enforced the hypothesis that estrogens promote cell viability and migration in vitro due to the function of E1SO4 as an estrogen reservoir for E2 production that promotes the IMC cells proliferation. Also, an increase in androgen secretion was associated with a reduction in cell viability. Finally, in vivo assays showed large tumor reduction. Hormone assays determined that high estrogen levels and the reduction of androgen levels promote tumor growth in Balb/SCID IMC mice. In conclusion, estrogen levels reduction may be associated with a good prognosis. Also, activation of AR by increasing androgen production could result in effective therapy for IMC because their anti-proliferative effect.

Upregulation of the Oct3/4 Network in Basal Breast Cancer Is Associated with Its Metastatic Potential and Shows Tissue Dependent Variability.

Adaptive plasticity of Breast Cancer stem cells (BCSCs) is strongly correlated with cancer progression and resistance, leading to a poor prognosis. In this study, we report the expression profile of several pioneer transcription factors of the Oct3/4 network associated with tumor initiation and metastasis. In the triple negative breast cancer cell line (MDA-MB-231) stably transfected with human Oct3/4-GFP, differentially expressed genes (DEGs) were identified using qPCR and microarray, and the resistance to paclitaxel was assessed using an MTS assay. The tumor-seeding potential in immunocompromised (NOD-SCID) mice and DEGs in the tumors were also assessed along with the intra-tumor (CD44+/CD24-) expression using flow cytometry. Unlike 2-D cultures, the Oct3/4-GFP expression was homogenous and stable in 3-D mammospheres developed from BCSCs. A total of 25 DEGs including Gata6, FoxA2, Sall4, Zic2, H2afJ, Stc1 and Bmi1 were identified in Oct3/4 activated cells coupled with a significantly increased resistance to paclitaxel. In mice, the higher Oct3/4 expression in tumors correlated with enhanced tumorigenic potential and aggressive growth, with metastatic lesions showing a >5-fold upregulation of DEGs compared to orthotopic tumors and variability in different tissues with the highest modulation in the brain. Serially re-implanting tumors in mice as a model of recurrence and metastasis highlighted the sustained upregulation of Sall4, c-Myc, Mmp1, Mmp9 and Dkk1 genes in metastatic lesions with a 2-fold higher expression of stem cell markers (CD44+/CD24-). Thus, Oct3/4 transcriptome may drive the differentiation and maintenance of BCSCs, promoting their tumorigenic potential, metastasis and resistance to drugs such as paclitaxel with tissue-specific heterogeneity.