Negative Breast Cancer Cells Research Articles

Targeted Destruction of S100A4 Inhibits Metastasis of Triple Negative Breast Cancer Cells.

Most patients who die of cancer do so from its metastasis to other organs. The calcium-binding protein S100A4 can induce cell migration/invasion and metastasis in experimental animals and is overexpressed in most human metastatic cancers. Here, we report that a novel inhibitor of S100A4 can specifically block its increase in cell migration in rat (IC50, 46 µM) and human (56 µM) triple negative breast cancer (TNBC) cells without affecting Western-blotted levels of S100A4. The moderately-weak S100A4-inhibitory compound, US-10113 has been chemically attached to thalidomide to stimulate the proteasomal machinery of a cell. This proteolysis targeting chimera (PROTAC) RGC specifically eliminates S100A4 in the rat (IC50, 8 nM) and human TNBC (IC50, 3.2 nM) cell lines with a near 20,000-fold increase in efficiency over US-10113 at inhibiting cell migration (IC50, 1.6 nM and 3.5 nM, respectively). Knockdown of S100A4 in human TNBC cells abolishes this effect. When PROTAC RGC is injected with mouse TNBC cells into syngeneic Balb/c mice, the incidence of experimental lung metastases or local primary tumour invasion and spontaneous lung metastasis is reduced in the 10-100 nM concentration range (Fisher's Exact test, p ≤ 0.024). In conclusion, we have established proof of principle that destructive targeting of S100A4 provides the first realistic chemotherapeutic approach to selectively inhibiting metastasis.

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.

Acute Exercise Increases NK Cell Mitochondrial Respiration and Cytotoxicity against Triple-Negative Breast Cancer Cells under Hypoxic Conditions.

Triple-negative breast cancer (TNBC) is an aggressive, highly metastatic malignancy with high recurrence rates. Hypoxia is a hallmark of the TNBC tumor microenvironment, which promotes tumor growth while impairing natural killer (NK) cell cytotoxic functions. Although acute exercise improves NK cell function under normoxic conditions, the effect of exercise on NK cell cytotoxic functions under hypoxic conditions mimicking O 2 tensions observed in solid tumors is unknown. The cytotoxic functions of resting and postexercise NK cells isolated from thirteen young inactive healthy women were assessed against breast cancer cells expressing different levels of hormone receptors (MCF-7 and MDA-MB-231) under normoxic and hypoxic conditions. Mitochondrial respiration and H 2 O 2 efflux rates of the TNBC-activated NK cells were assessed via high-resolution respirometry. Under hypoxia, postexercise NK cells exhibited greater killing of TNBC than resting NK cells. Further, postexercise NK cells were more likely to kill TNBC under hypoxia than normoxic conditions. In addition, mitochondrial respiration associated with oxidative (OXPHOS) capacity of TNBC-activated NK cells was greater in postexercise cells than resting cells under normoxia, but not under hypoxia. Finally, acute exercise was associated with reduced mitochondrial H 2 O 2 efflux by NK cells in both conditions. Together, we present crucial interrelationships between hypoxia and exercise-induced changes in NK cell functions against TNBC cells. By modulating their mitochondrial bioenergetic functions, we postulate that acute exercise improves NK cell function under hypoxic conditions. Specifically, NK cell O 2 and H 2 O 2 flow (pmol·s -1 ·million NK cells -1 ) changes in response to 30-min cycling suggest that exercise primes NK cell tumor killing by reducing mitochondrial oxidative stress and, thus, rescuing their function when exposed to harsh hypoxic environments as observed in the microenvironment of breast solid tumors.

Autophagy dictates sensitivity to PRMT5 inhibitor in breast cancer

Protein arginine methyltransferase 5 (PRMT5) catalyzes mono-methylation and symmetric di-methylation on arginine residues and has emerged as a potential antitumor target with inhibitors being tested in clinical trials. However, it remains unknown how the efficacy of PRMT5 inhibitors is regulated. Here we report that autophagy blockage enhances cellular sensitivity to PRMT5 inhibitor in triple negative breast cancer cells. Genetic ablation or pharmacological inhibition of PRMT5 triggers cytoprotective autophagy. Mechanistically, PRMT5 catalyzes monomethylation of ULK1 at R532 to suppress ULK1 activation, leading to attenuation of autophagy. As a result, ULK1 inhibition blocks PRMT5 deficiency-induced autophagy and sensitizes cells to PRMT5 inhibitor. Our study not only identifies autophagy as an inducible factor that dictates cellular sensitivity to PRMT5 inhibitor, but also unearths a critical molecular mechanism by which PRMT5 regulates autophagy through methylating ULK1, providing a rationale for the combination of PRMT5 and autophagy inhibitors in cancer therapy.

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.

Bovine milk-derived extracellular vesicles enhance doxorubicin sensitivity in triple negative breast cancer cells by targeting metabolism and STAT signalling.

Metastatic triple-negative breast cancer (TNBC) has a low 5-year survival rate of below 30% with systemic chemotherapy being the most widely used treatment. Bovine milk-derived extracellular vesicles (MEVs) have been previously demonstrated to have anti-cancer attributes. In this study, we isolated bovine MEVs from commercial milk and characterised them according to MISEV guidelines. Bovine MEVs sensitised TNBC cells to doxorubicin, resulting in reduced metabolic potential and cell-viability. Label-free quantitative proteomics of cells treated with MEVs and/or doxorubicin suggested that combinatorial treatment depleted various pro-tumorigenic interferon-inducible gene products and proteins with metabolic function, previously identified as therapeutic targets in TNBC. Combinatorial treatment also led to reduced abundance of various STAT proteins and their downstream oncogenic targets with roles in cell-cycle and apoptosis. Taken together, this study highlights the ability of bovine MEVs to sensitise TNBC cells to standard-of-care therapeutic drug doxorubicin, paving the way for novel treatment regimens.

3‐Methoxy‐Pyrrole‐Based Small Molecule Damages Mitochondria to Instigate Apoptosis in Breast Cancer Cells

AbstractMitochondrion has been implicated in the development and progression of breast cancer, making it an unconventional target for the anti‐cancer therapy. As a result, there is a need to explore novel small molecules for next generation breast cancer therapy. Towards this endeavour, herein, we have identified a novel 3‐methoxy‐pyrrole‐based small molecule from a synthetic library. Confocal fluorescence microscopy studies revealed that, this lead small molecule induced mitochondrial damage and generated reactive oxygen species (ROS) in the MCF7 breast cancer cells. Small molecule based mitochondrial impairment subsequently triggered cell cycle arrest and nuclear DNA damage followed by apoptosis leading remarkable cell killing and inhibition in the colony formation in MCF7 cells. Moreover, this 3‐methoxy‐pyrrole killed drug resistant MDA‐MB‐231 triple negative breast cancer cells and OVCAR8 ovarian cancer cells efficiently. This novel small molecule can open a new direction towards mitochondria targeted breast cancer therapy in future.

Preparation of antioxidant-biodegradable poly(acrylic acid-co-2-hydroxyethyl methacrylate) hydrogel using rutin as a crosslinker: Drug release and anticancer activity

It is important to develop drug-release systems that reduce the side effects of chemotherapy drugs, can regulate drug release, and can be designed for a target to keep the quality of life of cancer patients high. In this study, a polyphenol compound rutin was used as a natural crosslinker to prepare poly(acrylic acid-co-2-hydroxyethyl methacrylate)/rutin (RTN hydrogel) as a drug carrier. The swelling behavior, thermal stability, in vitro biodegradability, and antioxidant activity of this hydrogel were determined. The prepared hydrogel had in vitro biodegradability and antioxidant properties. The maximum doxorubicin (DOX) amount adsorbed by the RTN hydrogel was 32.96 ± 1.83 mg/g. The DOX-loaded RTN hydrogel displayed drug release behavior in normal and cancer cell media. At the same time, the effect of the DOX-loaded hydrogel on triple negative breast cancer (TNBC) cell lines was investigated. Our results show that the RTN hydrogel, prepared using the natural product of rutin as a crosslinker with in vitro biodegradability and antioxidant properties, offers great promise as a high-impact carrier in the drug distribution system.

Rational design of novel microtubule targeting anticancer drugs N-imidazopyridine noscapinoids: Chemical synthesis and experimental evaluation based on in vitro using breast cancer cells and in vivo using xenograft mice model

We present N-imidazopyridine-noscapinoids, a new class of noscapine derivatives that bind to tubulin and exhibit antiproliferative activity against triple positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells. The N-atom of the isoquinoline ring of noscapine scaffold was altered in silico by coupling the imidazo [(Ye et al., 1998; Ke et al., 2000) 1,21,2-a] pyridine pharmacophore to rationally develop a series of N-imidazopyridine-noscapinoids (7–11) with high tubulin binding affinity. The predicted ΔGbinding of the N-imidazopyridine-noscapinoids 7–11 varied from −27.45 to −36.15 kcal/mol, a much lower value than noscapine with ΔGbinding −22.49 kcal/mol. The cytotoxicity of N-imidazopyridine-noscapinoids was evaluated using hormone dependent MCF-7, triple negative MDA-MB-231 breast cancer cell lines and primary breast cancer cells. The cytotoxicity of these compounds (represented as IC50 concentration) ranges between 4.04 and 33.93 μM against breast cancer cells without affecting normal cells (IC50 value > 952 μM). All the compounds (7–11) perturbed the cell cycle progression at G2/M phase and triggered apoptosis. Among all the N-imidazopyridine-noscapinoids, N-5-Bromoimidazopyridine-noscapine (9) showed promising antiproliferative activity and was selected for detailed investigation. The onset of apoptosis treated with 9 using MDA-MB-231 revealed morphological changes like cellular shrinkage, chromatin condensation, membrane blebbing, and apoptotic bodies formation. Along with elevated reactive oxygen species (ROS), there was a loss of mitochondrial membrane potential, suggesting induction of apoptosis to cancer cells. Compound 9 was also found to significantly regress the implanted tumour in nude mice as xenografts of MCF-7 cells without any apparent side effects after drug administration. We conclude that N-imidazopyridine-noscapinoids possess excellent potential as a promising drug for treating breast cancers.

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.

Serum-Stable Gold(III) Bisphosphine Complex Induces Mild Mitochondrial Uncoupling and In Vivo Antitumor Potency in Triple Negative Breast Cancer.

The preparation of cyclometalated complexes offers a path to stable materials, catalysts, and therapeutic agents. Here, we explore the anticancer potential of novel biphenyl organogold(III) cationic complexes supported by diverse bisphosphine ligands, Au-1-Au-5, toward aggressive glioblastoma and triple negative breast cancer cells (TNBCs). The [C^C] gold(III) complex, Au-3, exhibits significant tumor growth inhibition in a metastatic TNBC mouse model. Remarkably, Au-3 displays promising blood serum stability over a relevant therapeutic window of 24 h and alteration in the presence of excess L-GSH. The mechanism-of-action studies show that Au-3 induces mitochondrial uncoupling, membrane depolarization, and G1 cell cycle arrest and prompts apoptosis. To the best of our knowledge, Au-3 is the first biphenyl gold-phosphine complex to uncouple mitochondria and inhibit TNBC growth in vivo.

Arsenic reduces the GATA3 expression associated with an increase in proliferation and migration of mammary epithelial cell line MCF-10A

Arsenic is associated with the development of breast cancer. However, the molecular mechanisms of arsenic induction of breast cancer are not fully defined. Interaction with zinc finger (ZnF) motifs in proteins is one of the proposed mechanisms of arsenic toxicity. GATA3 is a transcription factor that regulates the transcription of genes associated with cell proliferation, cell differentiation and the epithelial-mesenchymal transition (EMT) in mammary luminal cells. Given that GATA3 possesses two ZnF motifs essential for the function of this protein and that arsenic could alter the function of GATA3 through interaction with these structural motifs, we evaluated the effect of sodium arsenite (NaAsO2) on GATA3 function and its relevance in the development of arsenic-induced breast cancer. Breast cell lines derived from normal mammary epithelium (MCF-10A), hormone receptor-positive and hormone receptor negative breast cancer cells (T-47D and MDA-MB-453, respectively) were used. We observed a reduction on GATA3 protein levels at non-cytotoxic concentrations of NaAsO2 in MCF-10A and T-47D, but not in MDA-MB-453 cells. This reduction was associated with an increase in cell proliferation and cell migration in MCF-10A, but not in T-47D or MDA-MB-453 cells. The evaluation of cell proliferation and EMT markers indicate that the reduction on GATA3 protein levels by arsenic, disrupts the function of this transcription factor. Our data indicate that GATA3 is a tumor suppressor in the normal mammary epithelium and that arsenic could act as an initiator of breast cancer by disrupting the function of GATA3.

Extracellular mechanical forces and ferroptosis in triple negative breast cancer (TNBC): Targeting of aurora kinase A (AURKA).

e13086 Background: The mechanics of tumor microenvironment, primarily induced by extracellular matrix (ECM), function to regulate cell biological behaviors. However, it is unclear if and how triple negative breast cancer (TNBC) cells adapt their biological characteristics to variable extracellular mechanical forces. Methods: TNBC tissues of different stiffness from patients were harvested for untargeted metabolomics to investigate the potential effects of mechanical forces on metabolism. TNBC cells were cultured within gels of different type I collagen (Col-I) densities, which were the major component of ECM, to simulate the effects of extracellular mechanical forces on cells. The metabolic changes of TNBC cells were also examined by using untargeted metabolomics. TNBC cells bathed in different Col-I densities were injected subcutaneously into the breast pad of nude mice. Ferroptosis indicators were evaluated between different groups of mechanical force both in vitro and in vivo. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were carried out to examine the expression of ferroptosis-related genes and proteins between different groups of mechanical force. Results: Untargeted metabolomics studies showed significant differences in ferroptosis-related metabolites between TNBC tissues with different stiffness, which were also verified in TNBC cells subjected to different extracellular mechanical forces. In vitro experiment suggested that TNBC cells cultured within high Col-I density (strong extracellular forces) exhibited higher levels of lipid peroxidation, reactive oxygen accumulation, mitochondria membrane potential and iron concentration, and lower levels of total glutathione and reduced glutathione concentration, compared with low Col-I density (weak extracellular forces). In addition, mitochondria in high Col-I density group appeared to have volume decrease, fracture and fuzzy cristae. The above results indicated that extracellular mechanical force significantly induced ferroptosis in TNBC. RT-qPCR and western blot of ferroptosis-related genes and protein showed that both mRNA and protein levels of aurora kinase A (AURKA) were significantly decreased in TNBC cells with strong extracellular mechanical force. In addition, overexpression of AURKA inhibited ferroptosis caused by mechanical forces. Furthermore, this cause and effect among mechanical force, AURKA and ferroptosis was also evident in vivo experiments. Conclusions: Our data clarified the role of extracellular mechanical forces in TNBC and showed that the extracellular mechanical forces induced ferroptosis through decreasing the expression of AURKA.

The combination of APR-246 and carboplatin in olaparib-resistant high grade serous ovarian cancer (HGSOC) and triple negative breast cancer (TNBC) cell lines.

e17504 Background: PARP inhibitors (PARPi), as Olaparib (OLA), have changed the therapeutic landscape in HGSOC and TNBC. However, most patients will develop resistance to PARPi. Although resistance mechanisms are poorly understood, it is suggested that BRCA mutations reversions or cell cycle alterations could be involved. APR-246 is a new targeted agent that restores p53 in TP53-mutated cells. The aim of our study was to assess the potential synergism of the combination of carboplatin (carbo) plus APR-246 in both OLA-sensitive and OLA-resistant HGSOC and TNBC cell lines. Methods: We selected two HGSOC (PEO1-S and Kuramochi-S) and one TNBC TP53 mutant cell lines (MDA-MB-231-S). Resistance to OLA was generated by pulse doses with IC75 OLA for 24 hours and recovered for two weeks during a period of 6 months (PEO1-R) and to increased concentrations of OLA for 72 hours during a period of 1 year (Kuramochi-R, MDA-MB-231-R). Next-generation sequencing was performed in order to assess mutations. IC50 of APR-246 and Carbo and the combination of both (combo) were calculated for Kura-S/R, PEO1-S/R and 231-S/R cell lines. Synergism between both drugs was assessed with Chou-Talalay method for combination index (CI) at Fa50. DNA damage was assessed by gamma-H2AX expression by western-blot (WB). Results: 231-S harboured a TP53 mutation (mut) (missense R280K) and BRCA wildtype (wt), in PEO1-S BRCA2 mut p.Y1655* and TP53 mut was detectable. Kuramochi-S was TP53 mut (missense D816Y) and BRCA2 mut. BRCA and p53 mutation remained unchanged in the resistant lines generated.The mutation profile of each line and the IC50 of APR-246, carbo and combo are shown in the table. IC50 of carboplatin is superior in all 3 resistant cell lines when compared to their sensitive parental line, suggesting a potential cross-resistance to platinum in OLA-resistance. The combination of carbo and APR-246 is synergistic even in OLA-resistant cell lines, in fact in those cross-resistant lines values of synergism by Chou-Talalay are improved by the combo suggesting a potential role of this combination in PARPi resistant cell lines. WB showed an increased DNA damage with the combo when compared to either single agent, with an increase in H2AX in all lines either sensitive or resistant. Conclusions: The combination of APR246 and carboplatin is synergistic in all 3 studied cell lines but also in the OLA resistant ones. The combo increased its synergistic effect suggesting a potential role of combo in OLA-resistant tumors. [Table: see text]

Discovery of CDK4/6 bifunctional degraders for ER+/HER2- breast cancer.

1083 Background: CDK4/6 inhibitors (CDK4/6i) such as palbociclib and ribociclib are used to treat ER+/HER2- breast cancer, but patients can develop resistance via many mechanisms, several of which converge on the upregulation of CDK6. This has been shown to limit the effectiveness of CDK4/6i in ER+ breast cancer with up to 20% patients exhibiting innate resistance and up to 70% patients developing acquired resistance after 3 years on therapy (Scheidemann, 2021, doi:10.3390/ijms222212292). Methods: To address this limitation, we utilized our PRODEGY platform of Cereblon (CRBN) binders to synthesize CRBN mediated CDK4/6 bifunctional degraders to potently inhibit tumor growth for treatment of naïve ER+/HER2- breast cancer and CDK4/6i resistant tumors. Results: Target degradation by immunoblot analysis of the triple negative breast cancer (TNBC) cell line, MDA-MB-231, treated with our CDK4/6 bifunctional degraders for 6 hours showed up to 85% degradation of CDK4 and CDK6 with DC50s of 1-100nM. CDK4/6 phosphorylates the protein RB which releases the transcription factor E2F, inducing the expression of genes which promote cell cycle progression. Analysis of RB phosphorylation by in-cell western upon 24 hours of CDK4/6 degrader treatment showed phospho-RB IC50s at <30nM. Cell cycle analysis by staining with propidium iodide after 24 hours of treatment with CDK4/6 degraders induced G0/G1 cell cycle arrest at concentrations as low as 10nM. We used a 2D colony formation assay (CFA) as a readout for inhibition of proliferation by cell cycle arrest. Our CDK4/6 degraders showed potent inhibition of cell proliferation with CFA IC50s of <100nM in TNBC cell lines and <25nM in ER+ cell lines, including MCF7, T47D and ZR751 compared to CDK4/6i which ranged from 200nM to 500nM in MCF7 cells. We demonstrated that our CDK4/6 bifunctional degraders were significantly more potent in vitro than the CDK4/6i ribociclib and palbociclib, and the increased activity was due to CRBN mediated target degradation. Our CDK4/6 bifunctional degraders display excellent pharmacokinetic properties in mice with half-lives between 2-10 hours, oral bioavailability between 50-96%. MCF7 xenograft results with our proof-of-concept CDK4/6 degrader showed dose-dependent tumor growth inhibition and greater potency compared to the clinical CDK4/6i ribociclib. We saw tumor regression with our degrader at higher doses which we did not see at any dose of CDK4/6i. Conclusions: Our CDK4/6 bifunctional degraders display excellent single agent activity in vitro and in vivo particularly in comparison to clinically approved CDK4/6i, indicating that using a degrader approach to targeting this pathway may be more effective than current inhibitor therapies.

Influence of chromatin remodeling molecule ARID1A on metastatic heterogeneity in triple negative breast cancer: Binding of YAP.

1085 Background: Heterogeneity is the prominent character of TNBC and one predominant cause of treatment failure. As a key nuclear SWI/SNF protein complex subunit, ARID1A plays a critical role in tumor identity determining by manipulating gene expression. The present study aimed to investigate whether and how ARID1A determined the metastatic heterogeneity of triple-negative breast cancer. Methods: This study retrospectively collected and analysed clinical and pathological data from 258 patients diagnosed with triple-negative breast cancer at Fudan University Cancer Hospital from August 2015 to June 2016 to define the prognostic ability of ARID1A.CRISPR/Cas9 or plasmids-mediated ARID1A were used to determine the ARID1A function in regulating the metastatic character in TNBC. Cell morphology, tumor invasion, and metastasis were analyzed in terms of ARID1A status both in vitro and in vivo experiments. The molecular mechanism of ARID1A determining metastasis potential of TNBC was explored by RNA-Seq and nuclear positioning. Results: ARID1A low expression was found to have independent prognostic value for poor OS (43.9 vs 53.6 months, P=0.0001) and RFS (31.0 vs 45.5 months, P=0.0029) in TNBC patients. Both nuclear and cytoplasmic protein analysis and Immunofluorescent localization assay confirm that ARID1A recruits the Hippo pathway effector YAP into nucleus in human triple negative breast cancer cells. Next we designed the YAP truncator plasmid and confirmed by Co-Immunoprecipitation that ARID1A could competitively bind to the WW domain of YAP to form the ARID1A/YAP complex. Furthermore, down-regulation of ARID1A promoted migration and invasion both in human triple negative breast cancer cells and xenograft model through Hippo/YAP signaling axis. Conclusions: Our study provides new insights into the previously unrecognized role of ARID1A in TNBC metastasis, which might provide new therapeutic options for patients with TNBC.

Attenuative Effect of Diallyl Trisulfide on Caspase Activity in TNF-α-induced Triple Negative Breast Cancer Cells.

Diallyl trisulfide (DATS) has been shown to prevent and inhibit carcinogenesis in cancer cells. We have previously shown DATS's ability to decrease the percentage of viable cells, inhibit cell migration and modulate genes involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-B) and mitogen-activated protein kinase (MAPK) signaling. This study aimed to compare the efficacy of DATS in tumor necrosis factor alpha (TNF-α) induced MDA-MB-231 and MDA-MB-468 cells and investigate its role in cell-death signaling via cell cycle, flow cytometry, and caspase assay. DATS exhibit a time-dependent accumulation of G2/M phase cells in both cell lines, with higher effects in the MDA-MB-468 for all time points. DATS's ability to decrease the percentage of viable cells in both MDA-MB-231 and MDA-MB-468 cells was shown by a significant but slight increase of early and late apoptosis in the presence of DATS compared to control. Moreover, MDA-MB-468 cells showed more sensitivity to the DATS effect, evidenced by the higher percentage of apoptosis than MDA-MB-231 cells. The caspase studies showed a significant increase in caspase 3 and 8 activity in the presence of DATS, compared to control, in both cell lines. DATS showed no significant increase in caspase 9 activity in both cell lines compared to the control. DATS-induced apoptosis in human breast cancer cells is mediated, at least in part, by cell cycle arrest and caspase activity. These findings provide information for future studies into the role of DATS in TNBC therapy and prevention.

Engineered Extracellular Vesicles with Compound-Induced Cargo Delivery to Solid Tumors.

Efficient delivery of functional factors into target cells remains challenging. Although extracellular vesicles (EVs) are considered to be potential therapeutic delivery vehicles, a variety of efficient therapeutic delivery tools are still needed for cancer cells. Herein, we demonstrated a promising method to deliver EVs to refractory cancer cells via a small molecule-induced trafficking system. We generated an inducible interaction system between the FKBP12-rapamycin-binding protein (FRB) domain and FK506 binding protein (FKBP) to deliver specific cargo to EVs. CD9, an abundant protein in EVs, was fused to the FRB domain, and the specific cargo to be delivered was linked to FKBP. Rapamycin recruited validated cargo to EVs through protein-protein interactions (PPIs), such as the FKBP-FRB interaction system. The released EVs were functionally delivered to refractory cancer cells, triple negative breast cancer cells, non-small cell lung cancer cells, and pancreatic cancer cells. Therefore, the functional delivery system driven by reversible PPIs may provide new possibilities for a therapeutic cure against refractory cancers.

Local production of active vitamin D3 metabolites in breast cancer cells by CYP24A1 and CYP27B1

The role of vitamin D3 and its metabolites in cancer and especially as a treatment option has been widely disputed. Clinicians noting low serum 25-hydroxyvitamin D3 [25(OH)D3] levels in their patients, recommend vitamin D3 supplementation as a method of reducing the risk of cancer; however, data supporting this are inconsistent. These studies rely on systemic 25(OH)D3 as an indicator of hormone status, but 25(OH)D3 is further metabolized in the kidney and other tissues under regulation by several factors. This study examined if breast cancer cells also possess the ability to metabolize 25(OH)D3, and if so, whether the resulting metabolites are secreted locally; if this ability reflects ERα66 status; and if they possess vitamin D receptors (VDR). To address this question, estrogen receptor alpha (ERα) positive (MCF-7) and ERα negative (HCC38 and MDA-MB-231) breast cancer cell lines were examined for expression of ERα66, ERα36, CYP24A1, CYP27B1, and VDR as well as for local production of 24,25-dihydroxyvitamin D3 [24,25(OH)2D3] and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] after treatment with 25(OH)D3. The results showed that independent of ER status, breast cancer cells express the enzymes CYP24A1 and CYP27B1, which are responsible for converting 25(OH)D3 into its dihydroxylated forms. Moreover, these metabolites are produced at levels comparable to the levels observed in blood. They are positive for VDR, indicating that they can respond to 1α,25(OH)2D3, which can upregulate CYP24A1. These findings suggest that vitamin D metabolites may contribute to the tumorigenicity of breast cancer via autocrine and/or paracrine mechanisms.

Opto-microfluidic assisted synthesis of photo-protoporphyrin (pPP) conjugated to hollow gold-albumin hybrid nanoshells to enhance the efficiency of photodynamic therapy of triple negative breast cancer cells

IntroductionProtoporphyrin-IX (PpIX), a photosensitizer used in photodynamic therapy, has limitations due to its hydrophobicity, rapid photobleaching, and low absorption peak in the red region. These limitations make the use of PpIX less effective for photodynamic therapy treatments. In this study, we harnessed the power of microfluidic technology to manipulate the properties of PpIX and quickly synthesize albumin-based hybrid nanoshells with high reproducibility. Methods and materialTo begin with, we designed a microfluidic chip with SolidWorksⓇ software; then the chip was fabricated in Poly(methyl methacrylate) (PMMA) material using micromilling and thermal bonding. We synthesized PpIX-loaded CTAB micelles and subsequently transformed the PpIX structure into photo-protoporphyrin (PPP,) by opto-microfluidic chip (Integrating a microfluidic chip with a light source). Simultaneously with CTAB-PPP synthesis complex, we trapped it in binding sites of bovine serum albumin (BSA). Afterward, we used the same method (without irradiating) to generate a hybrid nanostructure consisting of hollow gold nanoshells (HGN) and BSACTAB-PPP. Then, after physical characterization of nanostructures, the photodynamic effects of the agents (HGNs, CTAB-PpIX, BSA-CTABPpIX, HGN-BSA-CTAB-PpIX, CTAB-PPP, BSA-CTAB-PPP, and HGNs-BSA-CTAB-PPP) were evaluated on MDA-MB-231 and 4T1 cells and the cytotoxic properties of the therapeutic agents after treatment for 24, 48, and 72 hours were investigated using MTT assay. Finally, we analyzed the findings using GraphPad Prism 9.0 software. ResultsResults revealed that the opto-microfluidic assisted synthesis of HGN-BSA-CTAB-PPP is highly efficient and reproducible, with a size of 120 nm, a zeta potential of -16 mV, and a PDI index of 0.357. Furthermore, the cell survival analysis demonstrated that the HGNBSA-CTAB-PPP hybrid nanostructure can significantly reduce the survival of MDA-MB-231 and 4T1 cancer cells at low radiation doses (< 10 J/cm2) when exposed to an incoherent light source due to its strong absorption peak at a wavelength of 670 nm. ConclusionThis research indicates that developing albumin-based multidrug hybrid nanostructures using microfluidic technology could be a promising approach to design more efficient photodynamic therapy studies.