BT474 Cell Lines Research Articles

Abstract 991: ONC201 & TR57 reversibly depletes mtDNA content & regulates mitochondrial biogenesis in BT-474, human breast cancer cells

Abstract Recent demonstration that mechanism of action of small molecule imipridone ONC201 could be mediated through mitochondrial targeting (Graves et al., 2019; Greer et al., Ishizawa et al., 2019), was confirmed by identification of mitochondrial caseinolythic protease (ClpP), as the only target of ONC201 and its chemical analog TR57 (Graves et al., 2019). We further studied direct effect of these drugs on mitochondrial functions and mtDNA distribution in human breast cancer cells. Using respirometry and confocal microscopy, we demonstrated dose- and time dependent suppression of mitochondria oxygen consumption and degradation of mtDNA in cultured human BT-474 cell lines, treated with ONC201 and TR57. Both drugs, used in this study, induced significant and reversible decrease in the rate of proliferation as well as the content of mtDNA within the mitochondria (> by 50%), which was released into the cytosol. Semi-quantitative analysis of confocal images of mitochondrial matrix (MTDR, red fluorescence) and mtDNA (SYBR Green-1, green fluorescence) demonstrated 30-35% release of mtDNA from mitochondria/mitochondrial fragments. Exposure to ONC201 (10µM) or TR57 (50nM) for 48 h decreased mtDNA content by 60% and 85%, respectively. Effect of these drugs was reversible and washout of drugs restored the rate of proliferation as well as mtDNA content in time-dependent manner. Within 24h of washout, the total cellular mtDNA reached almost 90% of initial level, demonstrating fast activation of mtDNA synthesis upon removal of toxins. RT-PCR approach to monitor mtDNA in the cytosol and mitochondria confirmed release of mtDNA into the cytosol and associated decrease of mtDNA within mitochondria/mitochondrial fragments, following ONC201 or TR57 treatment of BT-474 cells. Taken together, our data demonstrate that these drugs reversibly modulate and regulate intracellular mtDNA content, through ONC201 and TR57 dependent regulation of mitochondrial biogenesis in human breast cancer cells. In conclusion, ONC201 & TR57 dependent reversible inhibition of cell proliferation and decrease in mtDNA content allow suggesting drug-dependent regulation of mitochondrial biogenesis in these BCC. Our observation warrants new direction in the elucidation of the mechanism of action of these drugs and search for novel efficient anti-cancer drugs. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Margarita I. Kobyakova, Serazhutdin Abdullaev, Yana V. Evstratova, Artem Mishukov, Irina Odinokova, Lee M. Graves, Ekhson Holmuhamedov. ONC201 & TR57 reversibly depletes mtDNA content & regulates mitochondrial biogenesis in BT-474, human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 991.

UCP-2 inhibitor enhanced the efficacy of trastuzumab against HER2 positive breast cancer cells

PurposeThis study aimed to investigate the possibility of UCP-2 inhibitor in reducing acquired resistance of trastuzumab to improve the outcome of patients receiving trastuzumab therapy by exploring the relationship between UCP-2 expression and HER2 signaling pathway and examining whether UCP-2 expression was modulated by trastuzumab treatment.Methods32 women diagnosed with primary HER2-positive breast cancer were recruited in this study. Needle biopsy was obtained from patients before they received at least four cycles neoadjuvant therapy containing trastuzumab in combination with chemotherapy. Surgical tumor biopsy was obtained during surgical procedure after the neoadjuvant therapy. Levels of HER2 phosphorylation and UCP-2 expression were detected by immunohistochemistry (IHC) and compared between tumor needle biopsy tissue and surgical tumor samples of these patients, as well as in BT474 breast cancer cells before and after trastuzumab treatment. HER2-selective phosphorylation/kinase activity inhibitor ONT-380 was used to identify the correlation between HER2 phosphorylation level and UCP-2 expression. UCP-2 inhibitor Genipin was then used to evaluate the apoptosis index in BT474 cells treated with trastuzumab.ResultsUCP-2 expression was significantly elevated in surgical tumor samples from breast cancer patients receiving trastuzumab in a neoadjuvant setting. We further confirmed our findings in HER2-positive BT474 cell line and found that trastuzumab treatment induced phosphorylation of HER2 and the overexpression of UCP-2, and the latter can be reversed by HER2 selective kinase inhibitor ONT-380. Moreover, UCP-2 inhibitor Genipin significantly enhanced the proliferation suppression effects of trastuzumab and markedly promoted apoptosis.ConclusionTaken together, our study identified UCP-2 as a novel therapeutic target for HER2 positive breast cancer and UCP-2 inhibitor may have great potential to enhance the response rate and efficacy of trastuzumab therapy.

Soluble Cytoplasmic Expression and Purification of Immunotoxin HER2(scFv)-PE24B as a Maltose Binding Protein Fusion.

Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.

Increased FNDC5/IRISIN protein expression in breast cancer tissue is associated with obesity in postmenopausal women

AimTo analyse the fibronectin type III domain containing 5 (FNDC5)/irisin expression in tumour tissue of postmenopausal women presenting breast cancer and different body mass indexes (BMIs), proposing that obesity deregulates...

Cleavage of Syndecan-1 Promotes the Proliferation of the Basal-Like Breast Cancer Cell Line BT-549 Via Akt SUMOylation.

Basal-like breast cancer is characterized by an aggressive clinical outcome and presence of metastasis, for which effective therapies are unavailable. We have previously shown that chondroitin 4-O-sulfotransferase-1 (C4ST-1) controls the invasive properties of the basal-like breast cancer cell line BT-549 by inducing matrix metalloproteinase (MMP) expression through the N-cadherin/β-catenin pathway. Here we report that C4ST-1 controls the proliferation of BT-549 cells via the MMP-dependent cleavage of syndecan-1. Syndecan-1 is a membrane-bound proteoglycan associated with an aggressive phenotype and poor prognosis in breast cancer. In addition, the cleavage of syndecan-1 at a specific juxtamembrane cleavage site is implicated in the pathophysiological response in breast cancer. Knockout of C4ST-1 remarkably suppressed both the cleavage of syndecan-1 and proliferation of BT-549 cells. Kinases (AKT1, ERK1/2, PI3K, and STAT3) comprising cancer proliferative pathways are phosphorylated in C4ST-1 knockout cells at a level similar to that in parental BT-549 cells, whereas levels of phosphorylated S6 kinase and SUMOylated AKT (hyperactivated AKT observed in breast cancer) decreased in C4ST-1 knockout cells. An MMP inhibitor, GM6001, suppressed the small ubiquitin-like modifier (SUMO) modification of AKT, suggesting that cleavage of syndecan-1 by MMPs is involved in the SUMO modification of AKT. Forced expression of the cytoplasmic domain of syndecan-1, which is generated by MMP-dependent cleavage, increased the SUMO modification of AKT and global protein SUMOylation. Furthermore, syndecan-1 C-terminal domain-expressing BT-549 cells were more proliferative and sensitive to a potent SUMOylation inhibitor, tannic acid, compared with BT-549 cells transfected with an empty expression vector. These findings assign new functions to the C-terminal fragment of syndecan-1 generated by MMP-dependent proteolysis, thereby broadening our understanding of their physiological importance and implying that the therapeutic inhibition of syndecan-1 cleavage could affect the progression of basal-like breast cancer.

Down-regulated FST expression is involved in the poor prognosis of triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) is more commonly associated with young patients, featuring high histological grade, visceral metastasis, and distant recurrence. Follistatin (FST) is a secreted extracellular regulatory protein that antagonizes TGF-β superfamily such as activin and TGF-β related superfamily such as bone morphogenetic protein (BMP). The implication of FST in the proliferation, angiogenesis, and metastasis of solid tumors documents good or poor outcome of patients with BC. However, the role of FST in TNBC remains unclear.MethodsData of 935 patients with breast cancer (BC) were extracted from TCGA. Case–control study, Kaplan–Meier, uni-multivariate COX, and ROC curve were utilized to investigate the relationship between FST expression and the clinical characteristics and prognosis of BC. Functional studies were used to analyze the effect of FST expression on proliferation, apoptosis, migration, and invasion of TNBC cell lines. Bioinformatic methods such as volcanoplot, GO annd KEGG enrichment, and protein–protein interactions (PPI) analyses were conducted to further confirm the different roles of FST in the apoptotic pathways among mesenchymal and mesenchymal stem-like cells of TNBC.ResultsData from TCGA showed that low FST expression correlated with poor prognosis (for univariate analysis, HR = 0.47, 95% CI: 0.27–0.82, p = 0.008; for multivariate analysis, HR = 0.40, 95% CI: 0.21–0.75, p = 0.004). Low FST expression provided high predicted value of poor prognosis in TNBC amongst BCs. FST knockdown promoted the proliferation, migration and invasion of BT549 and HS578T cell lines. FST inhibited the apoptosis of mesenchymal cells by targeting BMP7.ConclusionsLow FST expression is associated with poor prognosis of patients with TNBC. FST expressions exhibit the anisotropic roles of apoptosis between mesenchymal and mesenchymal stem-like cells but promote the proliferation, migration, invasion in both of two subtypes of TNBC in vitro. FST may be a subtype-heterogeneous biomarker for monitoring the progress of TNBC.

In vitro cytotoxicity and neurotoxicity assessment of the alkaloidal constituents derived from Asimina triloba twigs

This study was aimed to evaluate the efficacy and safety of Asimina triloba extracts and its alkaloidal constituents in terms of their cytotoxicity and neurotoxicity towards different cell lines. Six alkaloids (anolobine-9-O-β-d-glucopyranoside, anolobine, norushinsunine, liriodenine, squamolone and coclaurine) were evaluated for their cytotoxic potential in four human solid tumor cell lines (SK-MEL, KB, BT-549, and SK-OV-3). The potential for neurotoxicity was determined using rat cortical neurons. Three extracts, namely a crude methanolic extract, an alkaloid-rich extract, and an acetogenin-rich extract, were also included in the study. The acetogenin-rich extract was the most potent, among all tested extracts, towards SK-MEL, KB, and BT-549 cells with IC50 values in the range of 0.09–0.11 µg/mL. Also, the same extract was found to be significantly toxic to the neurons at 10 µg/mL. The alkaloid-rich extract also showed cytotoxicity, albeit with lower potency, towards SK-MEL, KB, and BT-549 cell lines with IC50 values in the range of 1.7–2.6 µg/mL. Out of the six isolated alkaloids, three alkaloids (anolobine-9-O-β-D-glucopyranoside, norushinsunine, and liriodenine) demonstrated cytotoxic activity. Although the alkaloidal extract did not show any toxicity to neurons up to 10 µg/mL, the pure alkaloids were toxic to the neurons in a concentration-dependent manner. In conclusion, this study underlines the contribution of the alkaloidal constituents in the neurotoxicity and cytotoxicity of A. triloba.

RNA-Sequencing Reveals Heat Shock 70-kDa Protein 6 (HSPA6) as a Novel Thymoquinone-Upregulated Gene That Inhibits Growth, Migration, and Invasion of Triple-Negative Breast Cancer Cells.

ObjectiveBreast cancer has become the first highest incidence which surpasses lung cancer as the most commonly diagnosed cancer, and the second highest mortality among women worldwide. Thymoquinone (TQ) is a key component from black seed oil and has anti-cancer properties in a variety of tumors, including triple-negative breast cancer (TNBC).MethodsRNA-sequencing (RNA-seq) was conducted with and without TQ treatment in TNBC cell line BT-549. Gene Ontology (GO) function classification annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses for these genes were conducted. Western blot and semi-quantitative RT-PCR were used to verify the regulated gene. Functional assays by overexpression or knocking down were performed for HSPA6 and its mediator TQ for inhibiting growth, migration and invasion of TNBC cells. The regulatory mechanisms and prognosis for HSPA6 for breast cancer survival were conducted through bioinformatics and online databases.ResultsAs a result, a total of 141 downregulated and 28 upregulated genes were identified and 18 differentially expressed genes, which might be related to carcinomas, were obtained. Interestingly, GO and KEGG pathway showed their roles on anti-cancer and anti-virus. Further analysis found that the HSPA6 gene was the high significantly upregulated gene, and showed to inhibit TNBC cell growth, migration and invasion. High expression of HSPA6 was positively correlated with long overall survival (OS) in patients with breast cancer, indicating the tumor-suppressive roles for HSPA6. But DNA methylation of HSPA6 may not be the regulatory mechanism for HSPA6 mRNA upregulation in breast cancer tissues, although the mRNA levels of HSPA6 were increased in these cancer tissues compared with normal tissues. Moreover, TQ enhanced the inhibitory effect of migration and invasion when HSPA6 was overexpressed; while HSPA6 was knocked down, TQ attenuated the effects of HSPA6-promoted migration and invasion, demonstrating a partially dependent manner through HSPA6 by TQ treatment.ConclusionWe have successfully identified a novel TQ-targeted gene HSPA6, which shows the inhibitory effects on growth, migration and invasion in TNBC cells. Therefore, identification of HSPA6 not only reveals a new TQ regulatory mechanism, but also provides a novel candidate gene for clinical management and treatment of breast cancer, particularly for TNBC.

Identification of Serotonin as a Predictive Marker for Breast Cancer Patients.

PurposeCumulative evidence has demonstrated that breast cancer was the most commonly diagnosed cancer in women. Despite growing evidence for a link between serotonin and tumorigenesis, research on the expression of serotoninergic systems in the human breast cancer cell and tissue has only rarely been reported.MethodsFirst, immunofluorescence staining, ELISA and Western blotting were used to detect serotonin and melatoninergic systems in various breast cancer cell types. Then, serotonin expression was evaluated in the cultures of TPBC cell line BT-474 and TNBC cell line MDA-MB-231 using immunofluorescence assay. To further explore the diagnostic role of serotonin in breast cancer, serotonin expression was conducted in the TPBC and TNBC tumor sections by immunostaining analysis.ResultsOur results suggested that both human breast cancer cells and human breast epithelial cell line could synthesize serotonin and melatonin. Unlike melatonin, serotonin levels varied significantly between human breast cancer and breast epithelial cell line (p<0.01). In addition, serotonin N-acetyltransferase (NAT) and acetylserotonin methyltransferase (ASMT), the key enzymes in the pathway of melatonin synthesis from serotonin, were also detectable. In agreement with these findings of human breast cancer cell and human breast epithelial cell line, serotonin expression was also much higher in triple-negative (PR−, ER−, HER-2−) breast cancer (TNBC) and triple-positive breast cancer (TPBC) compared to para-carcinoma tissues (PCTs).ConclusionHere, we provided evidence that the human breast cancer cell (MCF-7, Bcap-37) and human breast epithelial cell (MCF-10A) could synthesize intrinsic serotonin and melatonin, and serotonin expression was higher in the breast cancer tissue compared with PCT. The findings suggested that serotonin might be used as a predictive marker for breast cancer patients.

In Vitro Cytotoxicity of Trastuzumab (Tz) and Se-Trastuzumab (Se-Tz) against the Her/2 Breast Cancer Cell Lines JIMT-1 and BT-474.

Her/2+ breast cancer accounts for ~25% mortality in women and overexpression of Her/2 leads to cell growth and tumor progression. Trastuzumab (Tz) with Taxane is the preferred treatment for Her/2+ patients. However, Tz responsive patients often develop resistance to Tz treatment. Herein, redox selenides (RSe-) were covalently linked to Tz using a selenium (Se)-modified Bolton–Hunter Reagent forming Seleno-Trastuzumab (Se-Tz; ~25 µgSe/mg). Se-Tz was compared to Tz and sodium selenite to assess the viability of JIMT-1 and BT-474 cells. Comparative cell viability was examined by microscopy and assessed by fluorometric/enzymatic assays. Se-Tz and selenite redox cycle producing superoxide (O2•−) are more cytotoxic to Tz resistant JIMT-1 and Tz sensitive BT-474 cells than Tz. The results of conjugating redox selenides to Tz suggest a wider application of this technology to other antibodies and targeting molecules.

Tailored-Made Polydopamine Nanoparticles to Induce Ferroptosis in Breast Cancer Cells in Combination with Chemotherapy.

Ferroptosis is gaining followers as mechanism of selective killing cancer cells in a non-apoptotic manner, and novel nanosystems capable of inducing this iron-dependent death are being increasingly developed. Among them, polydopamine nanoparticles (PDA NPs) are arousing interest, since they have great capability of chelating iron. In this work, PDA NPs were loaded with Fe3+ at different pH values to assess the importance that the pH may have in determining their therapeutic activity and selectivity. In addition, doxorubicin was also loaded to the nanoparticles to achieve a synergist effect. The in vitro assays that were performed with the BT474 and HS5 cell lines showed that, when Fe3+ was adsorbed in PDA NPs at pH values close to which Fe(OH)3 begins to be formed, these nanoparticles had greater antitumor activity and selectivity despite having chelated a smaller amount of Fe3+. Otherwise, it was demonstrated that Fe3+ could be released in the late endo/lysosomes thanks to their acidic pH and their Ca2+ content, and that when Fe3+ was co-transported with doxorubicin, the therapeutic activity of PDA NPs was enhanced. Thus, reported PDA NPs loaded with both Fe3+ and doxorubicin may constitute a good approach to target breast tumors.

Cell detachment ratio on pH-responsive chitosan: A useful biometric for prognostic judgment and drug efficacy assessment in oncology

The inherently unpredictable complexity of tumors impedes the widespread practice of the molecular biomarkers in outcome prediction. Alternatively, from the biophysical perspective, this study sought to investigate the applicability of the cell detachment ratio (CDR) derived from pH-responsive chitosan as a biometrical identifier for the disease state in cancer prognostic judgment and drug efficacy assessment. In the targeted therapy model, the repression of tumor dissemination in cells harboring aberrant ErbB signals (human non-small cell lung cancer cell line PC9 and breast cancer cell line BT474) were first demonstrated both in vitro and in vivo. Consequently, the corresponding CDR profile goes synchronously with the extent of cancer regression in response to the medication. Definitive integrins that drive the cell detachment were also verified through CDR examination following the integrin functional blockade. Conclusively, CDR is a promising clinical index for evaluation of the metastatic cell behaviors in terms of the cell detachment.

The Impact of Insulin on Low-dose Metronomic Vinorelbine and Mafosfamide in Breast Cancer Cells.

Breast cancer (BC) may be affected by diabetes and anti-diabetic medication, as well as its therapeutic agents. Low-dose metronomic chemotherapy (LDMC) is an available treatment option in BC. We investigated the impact of insulin on low-dose metronomic vinorelbine and mafosfamide in BC cell lines. Human BC cell lines T-47D, MCF-7, MDA-MB-231, BT-549 and non-tumorigenic breast cell line MCF-10A were exposed to 0.01 μg/ml and 10 μg/ml insulin in combination with low-dose metronomic vinorelbine or mafosfamide. The cell viability was determined after 24-72 hours using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Insulin, especially at a concentration of 10 μg/ml, seemed to increase viability of vinorelbine-treated hormone receptor-positive BC cells, whereas low-dose mafosfamide treatment tended to be potentiated by insulin in triple-negative cells. Our findings suggest that insulin may influence the cytotoxic activity of LDMC depending on insulin concentration, type of cytotoxic drug used and BC cell line.

The epithelial sodium channel has a role in breast cancer cell proliferation.

Breast cancer is the most common cancer affecting women worldwide with half a million associated deaths annually. Despite a huge global effort, the pathways of breast cancer progression are not fully elucidated. Ion channels have recently emerged as novel regulators of cancer cell proliferation and metastasis. The epithelial sodium channel, ENaC, made up of α, β and γ subunits is well known for its role in Na+ reabsorption in epithelia, but a number of novel roles for ENaC have been described, including potential roles in cancer. A role for ENaC in breast cancer, however, has yet to be described. Therefore, the effects of ENaC level and activity on breast cancer proliferation were investigated. Through the publicly available SCAN-B dataset associations between αENaC mRNA expression and breast cancer subtypes, proliferation markers and epithelial-mesenchymal transitionmarkers (EMT) were assessed. αENaC expression, through overexpression or siRNA-mediated knockdown, and activity, through the ENaC-specific inhibitor amiloride, were altered in MCF7, T47D, BT549, and MDAMB231 breast cancer cells. MTT and EdU cell proliferation assays were used to determine the effect of these manipulations on breast cancer cell proliferation. High αENaC mRNA expression was associated with less aggressive and less proliferative breast cancer subtypes and with reduced expression of proliferation markers. Decreased αENaC expression or activity, in the mesenchymal breast cancer cell lines BT549 and MDAMB231, increased breast cancer cell proliferation. Conversely, increased αENaC expression decreased breast cancer cell proliferation. αENaC expression is associated with a poor prognosis in breast cancer and is a novel regulator of breast cancer cell proliferation. Taken together, these results identify ENaC as a potential future therapeutic target.

Abstract PS19-28: Thymoquinone and tamoxifen co-treatment synergistically inhibit proliferation, invasion and induce apoptosis in human breast cancer cell lines in vitro and in vivo

Abstract Epidemiological studies and experimental analysis indicate that dietary factors influence thedevelopment of breast cancer, suggesting the role of natural products as modifying factors againstbreast cancer. Thymoquinone (TQ) (a dietary phytochemical compound) the main active ingredientof the volatile oil of black seed (Nigella sativa) has been used to be safe when administered to awide variety of normal cells. In addition, Tamoxifen (TAM), a nonsteroidal triphenylethylene derivateand selective ER modulator, has been used as a single agent in the treatment of ER positive breastcancer.In the present study, we report the possible chemo-sensitizing effect of TQ as a promisingconventional chemotherapeutic agent, using a panel of human breast cancer cell lines, MCF-7, ZR-75-1, T-47D and BT-20 respectively in vitro and in vivo.Our data revealed that TQ, TAM or combined treatments significantly inhibited cell viability of MCF-7, T-47D, ZR-75-1 but slightly of BT-20 cell lines consistently with down-regulation of signaltransducer and activator of transcription 3 (STAT 3), mTOR, cyclin D1, oncogenic β-catenin,signaling a specific transcription factor and PPAR-gamma in a dose dependent manner. Mostimportantly, combined treatment enhanced inhibition of cell viability, DNA fragmentation andapoptosis induction through Caspase-3 activation and Bcl-2 down regulation in all tested cell linesbut this effect was limited in BT-20 cells compared to controls. Moreover, our data was supported bymicroscopic examination using electron scanning microscopy that revealed many apoptoticalterations after combined treatment compared to each TQ or TAM. Furthermore, confocalfluorescence microscopy examination revealed that only combined treatment showed a significantinhibition of filopodia formation as a sign of invasion inhibition in both BT-20 and MCF-7 humanbreast cancer cell lines. In vivo, tumor xenotransplants of human ZR-75-1 cells revealed that onlycombined treatment showed a significant regression in tumor size by 53% after 72h and enhancedE-cadherin expression as analyzed by immunohistochemistry and confocal fluorescencemicroscopy.Taken together, our findings provide strong in vitro and in vivo molecular evidences in support ofour hypothesis that Thymoquinone synchronized as chemo-preventive agent with Tamoxifen to inhibithuman breast cancer cell lines proliferation, invasion and induce apoptosis that might have a potentialimplication in breast cancer prevention and treatment. Citation Format: Islam Yahiya, Heba Helmy, Ahmed Sultan. Thymoquinone and tamoxifen co-treatment synergistically inhibit proliferation, invasion and induce apoptosis in human breast cancer cell lines in vitro and in vivo [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-28.

Abstract PS11-17: Combination PI3K and NOS targeted therapy for metaplastic breast cancer

Abstract Metaplastic breast cancer (MpBC) is a rare subset accounting for less than 1% of all breast cancers. MpBC exhibits the worst prognosis in comparison to triple-negative breast cancer (TNBC) with a dismal poor survival rate in patients with metastatic disease. The main therapeutic options for MpBC remain surgery and systemic chemotherapy, despite known resistance to most systemic chemotherapy. A common molecular alteration in MpBC is hyperactivation of the phosphoinositide 3-kinase (PI3K) pathway. Recently, we published that MpBC displays a gain-of-function oncogenic mutation in ribosomal protein L39 (RPL39), which is responsible for treatment resistance, stem cell self-renewal, and lung metastasis. The mechanistic function of RPL39 is mediated through inducible nitric oxide synthase (iNOS)-mediated nitric oxide production. In addition, we demonstrated that inhibiting this nitric oxide synthase (NOS) pathway using pan-NOS inhibitor NG-methyl-L-arginine acetate (L-NMMA) may represent a highly effective therapeutic option for MBC patients. Therefore, we hypothesize that the combinatorial approach of inhibiting the two major pathways implicated in MpBC, namely PI3K/Akt/mTOR and NOS pathways would lead to significant tumor regression. Alpelisib, an FDA-approved, isoform-specific PI3K inhibitor, is currently used with the antiestrogen, fulvestrant, to treat hormone receptor (HR)-positive, PIK3CA-mutated breast cancer patients. We used MpBC cell lines Hs578t and BT549 and MpBC patient-derived xenograft (PDX) models in our preliminary studies. Using flow cytometry to detect Annexin V+/DAPI+ cells, we found increased cell death in MpBC cell lines treated with L-NMMA+alpelisib combination treatment in comparison to monotherapy. Immunoblotting of samples from single (L-NMMA or alpelisib) or combination treated cell lines and PDXs showed increased PARP degradation and cleaved caspase 3/9 with combination treatment. In vivo data using PDX models showed that combination treatment of L-NMMA+alpelisib was most effective at reducing tumor volume in comparison to monotherapy. We performed preliminary bulk-RNA sequencing analysis of tumor samples collected from PDX BCM-4664 treated with vehicle control, monotherapy of L-NMMA or alpelisib, and combination therapy. Gene set enrichment analysis found that E2F and Hedgehog signaling pathways were the top two enriched pathways in BCM-4664 tumors treated with combination therapy. Pathway-focused RT-PCR of MpBC PDXs confirmed GSEA results and showed significant gene expression alterations involving E2F signaling and cell cycle regulation. Further studies are currently ongoing to elucidate the molecular mechanisms involved in enhanced cell death and decreased tumorigenesis with L-NMMA and alpelisib dual therapy. Our results support the concept that LNMMA and alpelisib combination therapy has therapeutic potential in the treatment of MpBC, which may enable rapid translational into clinical trials, and impact the exceeding poor prognosis of women with MpBC. Citation Format: Tejaswini Parlapalle Reddy, Roberto R Rosato, Liliana Guzman, Wei Qian, Jianying Zhou, Hongbin Wang, Helen Piwnica-Worms, Stacy Moulder, Jenny C Chang. Combination PI3K and NOS targeted therapy for metaplastic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-17.

Abstract PR001: Cancer stem cells as origin of tumor associated immune cells

Abstract Development of cancer stem cell (CSC) models from induced pluripotent stem cells (iPSCs) for different types of cancers will help to investigate the tumorigenesis and tumor microenvironment heterogeneity. In this context, our lab have developed a novel method to generate CSCs from iPSCs under cancer microenvironment using conditioned medium from different cancer cell lines. On the other hand, the ability of CSCs to exhibit different hematopoietic cells is still unclear. In our previous study, BT549 cell line conditioned media was used to obtain CSCs. CSCs (miPSCs-BT) converted from mouse iPSCs were tumorigenic and expressed stemmens and cancer stem cell markers. CSCs converted from miPSCs and injected into mice were used. CSCs isolated from malignant tumor were cultured in absence of Leukemia Inhibitory Factor(LIF) where undifferentiated cells were selected using puromycin. Non-adherent cells (NACs) arising from adherent CSCs collected and their viability, the morphology and the expression of hematopoietic cell markers were investigated. NACs were also injected into the tail vein of busulfan conditioned Balb/c nude mice. CSCs were also induced to differentiate into macrophages while using IL3 and SCF. The round nucleated NACs were viable, positive for hematopoietic lineage markers and had CD34 positive subpopulation as well as the ability of homing to the bone marrow. Moreover, after 4 weeks of injection, Wright-Giemsa staining showed that the number of white blood cells was higher in injected mice with NACs comparing to the control. CSCs also showed the ability to differentiate toward macrophages. In conclusion, CSCs were demonstrated to have the potential to provide progenies with hematopoietic markers, morphology, and homing ability to the bone marrow, which could give new insight into the tumor microenvironment according to the plasticity of CSCs. This abstract is also being presented as PO057. Citation Format: Ghmkin Hassan, Said M. Afify, Akimasa Seno, Masaharu Seno. Cancer stem cells as origin of tumor associated immune cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PR001.

Comparative Evaluation of Novel 177Lu-Labeled PNA Probes for Affibody-Mediated PNA-Based Pretargeting.

Simple SummaryAffibody molecules are small, engineered affinity proteins based on a nonimmunoglobulin scaffold. Affibody-based radionuclide imaging probes have demonstrated excellent tumor targeting. However, the renal clearance of affibody molecules is accompanied by high reabsorption and retention of activity in the kidney, which prevents their use for radionuclide therapy. We have previously shown the feasibility of overcoming the high renal uptake using a pretargeting approach for affibody-mediated therapy based on peptide nucleic acid (PNA) hybridization. In this study, we test the hypothesis that shortening the PNA pretargeting probes would further increase the difference between the accumulation of radiometals in tumor xenografts and in kidneys. A series of novel PNA probes has been designed and evaluated in vitro and in vivo. We have found that a variant containing 9 nucleobases enables a two-fold increase of the tumor-to-kidney dose ratio compared with a variant containing 15 nucleobases. This creates preconditions for more efficient therapy of cancer. Affibody-mediated PNA-based pretargeting is a promising approach to radionuclide therapy of HER2-expressing tumors. In this study, we test the hypothesis that shortening the PNA pretargeting probes would increase the tumor-to-kidney dose ratio. The primary probe ZHER2:342-SR-HP15 and the complementary secondary probes HP16, HP17, and HP18, containing 9, 12, and 15 nucleobases, respectively, and carrying a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) chelator were designed, synthesized, characterized in vitro, and labeled with 177Lu. In vitro pretargeting was studied in HER2-expressing SKOV3 and BT474 cell lines. The biodistribution of these novel probes was evaluated in immunodeficient mice bearing SKOV3 xenografts and compared to the previously studied [177Lu]Lu-HP2. Characterization confirmed the formation of high-affinity duplexes between HP15 and the secondary probes, with the affinity correlating with the length of the complementary PNA sequences. All the PNA-based probes were bound specifically to HER2-expressing cells in vitro. In vivo studies demonstrated HER2-specific uptake of all 177Lu-labeled probes in xenografts in a pretargeting setting. The ratio of cumulated radioactivity in the tumor to the radioactivity in kidneys was dependent on the secondary probe’s size and decreased with an increased number of nucleobases. The shortest PNA probe, [177Lu]Lu-HP16, showed the highest tumor-to-kidney ratio. [177Lu]Lu-HP16 is the most promising secondary probe for affibody-mediated tumor pretargeting.

Design, synthesis and biological evaluation of water soluble and non-aggregated silicon phthalocyanines, naphthalocyanines against A549, SNU-398, SK-MEL128, DU-145, BT-20 and HFC cell lines as potential anticancer agents

Cancer has become an important public problem in worldwide since cancer incidence and mortality are growing rapidly. In this study, water soluble and non-aggregated silicon (IV) phthalocyanines and naphthalocyanines containing (3,5-bis{3-[3-(diethylamino)phenoxy]propoxy}phenyl)methoxy groups have been synthesized and characterized to investigate their anticancer potential. Their DNA binding/nuclease, topoisomerases inhibition were investigated using UV–Vis absorption, thermal denaturation and agarose gel electrophoresis. The in vitro cytotoxic properties of the compounds on human lung (A549), breast (BT-20), liver (SNU-398), prostate (DU-145), melanoma (SK-Mel 128) carcinoma and human fibroblast (HFC) normal cell lines were evaluated by using MTT assay. In order to determine the mechanism of cancer cell growth suppression, cell cycle analysis was carried out using flow cytometer on A549 cell line. The Kb values of SiPc1a and SiNc2a were 6.85 ± (0.35) × 106 and 1.72 ± (0.16) × 104 M−1 and Tm values of ct-DNA were calculated as 82.02 °C and 78.07 °C, respectively in the presence of both compounds. The ΔTm values of SiPc1a and SiNc2a were calculated as 6.45 and 2.50 °C, respectively. The nuclease effects of SiPc1a and SiNc2a with supercoiled plasmid pBR322 DNA demonstrated that both compounds did not trigger any DNA nuclease effects at the lowest concentrations without irradiation whereas both compounds in the presence of activating agent (H2O2) showed significant plasmid DNA nuclease actions under irradiation (22.5 J/cm2). SiPc1a and SiNc2a inhibited to topoisomerase I on increasing concentrations whilst they had lower inhibition action toward topoisomerase II that of topoisomerase I. The in vitro cytotoxicity studies displayed that SiPc1a had the highest cytotoxic effects among the tested compounds against A549, SNU-398, SK-MEL128, DU-145, BT-20 and HFC cell lines with CC50 values ranged from 0.49 to 2.99 µM. Furthermore, SiPc1a inhibited cell proliferation by cell cycle arrest in G0/G1 phase. All of these results suggested that SiPc1a is a promising candidate as an anticancer agent.

Synthesis, antitumor, antibacterial and urease inhibitory evaluation of new piperazinyl N-4 carbamoyl functionalized ciprofloxacin derivatives.

Quinolones are well known antibacterial chemotherapeutics. Furthermore, they were reported for other activities such as anticancer and urease inhibitory potential. Modification at C7 of quinolones can direct these compounds preferentially toward target molecules. Different derivatives of ciprofloxacin by functionalization at the piperazinyl N-4 position with arylidenehydrazinecarbonyl and saturated heterocyclic-carbonyl moieties have been synthesized and characterized using different spectral and analytical techniques. The synthesized compounds were evaluated for anticancer, antibacterial, and urease inhibitory activities. Among the synthesized compounds derivatives 3f and 3gexperienced a potent antiproliferative activity against the breast cancer BT-549 cell line, recording growth percentages of 28.68%and 6.18%, respectively. Additionally, compound 3g revealed a remarkable antitumor potential toward the colon cancer HCT-116 cells (growth percentage 14.76%). Activity of compounds3f and 3g against BT-549 cells was comparable to doxorubicin (IC50 = 1.84, 9.83, and 1.29µM, respectively). Test compounds were less active than their parent drug, ciprofloxacin toward Klebsiella pneumoniae and Proteus mirabilis. However, derivative 4a showed activity better than chloramphenicol against Klebsiella pneumoniae (MIC = 100.64 and 217.08µM, respectively). Meanwhile, many of the synthesized compounds revealed a urease inhibitory activitygreaterthantheir parent. Compound 3iwas the most potent urease inhibitorwith IC50 of 58.92µM, greater than ciprofloxacin and standard inhibitor, thiourea (IC50 = 94.32 and 78.89µM, respectively). This study provided promising derivatives as lead compounds for development of anticancer agents against breast and colon cancers, and others for optimization of urease inhibitors.