pyrimidinones and Triple-Negative-Breast-Neoplasms

Adavosertib (AZD1775) is a first-in-class, selective, small-molecule inhibitor of Wee1.. The safety, tolerability, pharmacokinetics, and efficacy of adavosertib monotherapy were evaluated in patients with various solid-tumor types and molecular profiles.. Eligible patients had the following: confirmed diagnosis of ovarian cancer (OC), triple-negative breast cancer (TNBC), or small-cell lung cancer (SCLC); previous treatment for metastatic/recurrent disease; and measurable disease. Patients were grouped into six matched cohorts based on tumor type and presence/absence of biomarkers and received oral adavosertib 175 mg twice a day on days 1-3 and 8-10 of a 21-day treatment cycle.. Eighty patients received treatment in the expansion phase; median total treatment duration was 2.4 months. The most common treatment-related adverse events (AEs) were diarrhea (56.3%), nausea (42.5%), fatigue (36.3%), vomiting (18.8%), and decreased appetite (12.5%). Treatment-related grade ≥ 3 AEs and serious AEs were reported in 32.5% and 10.0% of patients, respectively. AEs led to dose interruptions in 22.5%, reductions in 11.3%, and discontinuations in 16.3% of patients. One patient died following serious AEs of deep vein thrombosis (treatment related) and respiratory failure (not treatment related). Objective response rate, disease control rate, and progression-free survival were as follows: 6.3%, 68.8%, 4.5 months (OC BRCA wild type); 3.3%, 76.7%, 3.9 months (OC BRCA mutation); 0%, 69.2%, 3.1 months (TNBC biomarker [CCNE1/MYC/MYCL1/MYCN] non-amplified [NA]); 0%, 50%, 2 months (TNBC biomarker amplified); 8.3%, 33.3%, 1.3 months (SCLC biomarker NA); and 0%, 33.3%, 1.2 months (SCLC biomarker amplified).. Adavosertib monotherapy was tolerated and demonstrated some antitumor activity in patients with advanced solid tumors.. ClinicalTrials.gov identifier NCT02482311; registered June 2015.

Topics: Female; Humans; Lung Neoplasms; Ovarian Neoplasms; Pyrazoles; Pyrimidinones; Small Cell Lung Carcinoma; Triple Negative Breast Neoplasms

We report results from a phase II study assessing the efficacy of the WEE1 inhibitor adavosertib with cisplatin in metastatic triple-negative breast cancer (mTNBC).. Patients with mTNBC treated with 0-1 prior lines of chemotherapy received cisplatin 75 mg/m. A total of 34 patients initiated protocol therapy; median age was 56 years, 2 patients (6%) had. Among patients with mTNBC treated with 0-1 prior lines, adavosertib combined with cisplatin missed the prespecified ORR cutoff of >30%. The finding of immune-infiltrated tumors in patients with clinical benefit warrants validation.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle Proteins; Chemotherapy, Adjuvant; Cisplatin; Female; Humans; Middle Aged; Progression-Free Survival; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidinones; Triple Negative Breast Neoplasms; Young Adult

This study aimed to determine the safety, tolerability, and recommended phase II doses of trametinib plus uprosertib (GSK2141795) in patients with solid tumors likely to be sensitive to MEK and/or AKT inhibition.. This was a phase I, open-label, dose-escalation, and dose-expansion study in patients with triple-negative breast cancer or BRAF-wild type advanced melanoma. The primary outcome of the expansion study was investigator-assessed response. Among 126 enrolled patients, 63 received continuous oral daily dosing of trametinib and uprosertib, 29 received various alternative dosing schedules, and 34 were enrolled into expansion cohorts. Doses tested in the expansion cohort were trametinib 1.5 mg once daily (QD) + uprosertib 50 mg QD.. Adverse events (AEs) were consistent with those reported in monotherapy studies but occurred at lower doses and with greater severity. Diarrhea was the most common dose-limiting toxicity; diarrhea and rash were particularly difficult to tolerate. Overall, 59% and 6% of patients reported AEs with a maximum severity of grade 3 and 4, respectively. Poor tolerability prevented adequate delivery of uprosertib with trametinib at a concentration predicted to have clinical activity. The study was terminated early based on futility in the continuous-dosing expansion cohorts and a lack of pharmacological or therapeutic advantage with intermittent dosing. The objective response rate was < 5% (1 complete response, 5 partial responses).. Continuous and intermittent dosing of trametinib in combination with uprosertib was not tolerated, and minimal clinical activity was observed in all schedules tested.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Cohort Studies; Diamines; Dose-Response Relationship, Drug; Female; Follow-Up Studies; Humans; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Maximum Tolerated Dose; Melanoma; Middle Aged; Prognosis; Proto-Oncogene Proteins c-akt; Pyrazoles; Pyridones; Pyrimidinones; Tissue Distribution; Triple Negative Breast Neoplasms; Young Adult

Targeting the dysregulated BRAF-MEK-ERK pathway in cancer has increasingly emerged in clinical trial design. Despite clinical responses in specific cancers using inhibitors targeting BRAF and MEK, resistance develops often involving nongenomic adaptive bypass mechanisms. Inhibition of MEK1/2 by trametinib in patients with triple-negative breast cancer (TNBC) induced dramatic transcriptional responses, including upregulation of receptor tyrosine kinases (RTK) comparing tumor samples before and after one week of treatment. In preclinical models, MEK inhibition induced genome-wide enhancer formation involving the seeding of BRD4, MED1, H3K27 acetylation, and p300 that drives transcriptional adaptation. Inhibition of the P-TEFb-associated proteins BRD4 and CBP/p300 arrested enhancer seeding and RTK upregulation. BRD4 bromodomain inhibitors overcame trametinib resistance, producing sustained growth inhibition in cells, xenografts, and syngeneic mouse TNBC models. Pharmacologic targeting of P-TEFb members in conjunction with MEK inhibition by trametinib is an effective strategy to durably inhibit epigenomic remodeling required for adaptive resistance.

Topics: Animals; Antineoplastic Agents; Azepines; Cell Cycle Proteins; Cell Line, Tumor; Discoidin Domain Receptor 1; DNA Methylation; Drug Resistance, Neoplasm; Drug Synergism; Enhancer Elements, Genetic; Epigenesis, Genetic; Female; Heterocyclic Compounds, 4 or More Rings; Humans; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mice, Inbred BALB C; Mice, SCID; Molecular Targeted Therapy; Nuclear Proteins; Positive Transcriptional Elongation Factor B; Pyridones; Pyrimidinones; RNA Interference; Transcription Factors; Triazoles; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Breast cancer (BC) is a heterogeneous disease with different intrinsic subtypes. The most aggressive subtype of BC-triple-negative breast cancer (TNBC) is characterized by high heterogeneity and metastasis rate, poor prognosis and lack of therapeutic targets due to the absence of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2. Targeted therapies have been approved for many other cancers and even other subtypes of BC, but treatment options for TNBC are still mainly limited to chemotherapy. Therefore, new, more effective treatment regimens are needed. Combined chemotherapy with two or more active agents is considered a promising anti-neoplasm tool in order to achieve better therapeutic response and reduce therapy-related adverse effects. The study demonstrated an antagonistic effect commonly used in TNBC therapy cytostatic drug-paclitaxel (PAX) and sirtuin inhibitor: cambinol (CAM) in BT-549, MDA-MB-468 and HCC1937 TNBC cell lines. The type of pharmacological interaction was determined by a precise and rigorous pharmacodynamic method-isobolographic analysis. The cytotoxic and anti-proliferative effects of CAM used alone or combined with PAX were determined utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-bromo-2'-deoxyuridine (BrdU) assays, respectively. Induction of apoptosis in TNBC cell lines after PAX and CAM treatment applied individually or in combination was determined by flow cytometry (FACS) as a number of cells with active caspase-3. It has been observed that both agents used separately inhibit cell proliferation and induce apoptosis; however, applying them in combination ameliorated antiproliferative and pro-apoptotic effects in all analyzed TNBC cell lines. Our results demonstrate that CAM and PAX used in combination act antagonistically, limiting anti-cancer efficacy and showing the importance of preclinical testing.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Humans; Naphthalenes; Paclitaxel; Pyrimidinones; Sirtuins; Triple Negative Breast Neoplasms

Despite the continuously growing repertoire of new and improved anti-cancer therapies, triple-negative breast cancer (TNBC) remains a clinical challenge to treat. In this sense, targeting signaling pathways such as Notch and Wnt/β-catenin have attracted growing attention. This work aimed at investigating the possible antitumor effects of IMR-1 as a Notch inhibitor, PRI-724 as a Wnt/β-catenin inhibitor, as well as their combination and to explore the possible crosstalk between Notch and Wnt/β-catenin signaling pathways in MDA-MB-231 TNBC cell line. Microculture tetrazolium test (MTT) was used to determine the drug growth inhibition (GI50), and the results were analyzed using CompuSyn 3.0.1 software. MDA-MB-231 cells were divided into four treatment groups including positive control, IMR-1-treated, PRI-724-treated, and combination-treated groups. Sandwich enzyme-linked immunosorbent assay (ELISA) was used for the determination of the protein levels of hairy and enhancer of split-1 (HES-1), Notch-1, β-catenin, cyclin-D1, and vascular endothelial growth factor (VEGF1). HES-1 gene expression was assessed by quantitative real-time polymerase chain reaction. Statistical analyses were performed using GraphPad Prism Software. The GI50 for IMR-1 and PRI-724 were 15.3 μM and 0.69 μM, respectively. Upon treatment of MDA-MB-231 cells with these drugs, HES-1 gene expression was up-regulated due to single and combined treatments. Moreover, the protein levels of cyclin-D1, VEGF1, HES-1, and Notch-1 were reduced, while those of active β-catenin and active caspase-3 were elevated. IMR-1/PRI-724 combination augmented IMR-1- and PRI-724-mediated effects on MDA-MB-231 cells by initiating apoptotic cell death. Further in vitro and in vivo studies are warranted to support our findings.

Topics: Antineoplastic Agents; Apoptosis; beta Catenin; Bridged Bicyclo Compounds, Heterocyclic; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Drug Interactions; Humans; Pyrimidinones; Receptor, Notch1; Thiazolidines; Transcription Factor HES-1; Triple Negative Breast Neoplasms; Vascular Endothelial Growth Factor A; Wnt Signaling Pathway

Triple-negative breast cancers (TNBCs) represent 15% to 20% of all breast cancers and are often associated with poor prognosis. The lack of targeted therapies for TNBCs contributes to higher mortality rates. Aberrations in the phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase pathways have been linked to increased breast cancer proliferation and survival. It has been proposed that these survival characteristics are enhanced through compensatory signaling and crosstalk mechanisms. While the crosstalk between PI3K and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways has been characterized in several systems, new evidence suggests that MEK5/ERK5 signaling is a key component in the proliferation and survival of several aggressive cancers. In this study, we examined the effects of dual inhibition of PI3K/protein kinase B (Akt) and MEK5/ERK5 in the MDA-MB-231, BT-549, and MDA-MB-468 TNBC cell lines. We used the Akt inhibitor ipatasertib, ERK5 inhibitors XMD8-92 and AX15836, and the novel MEK5 inhibitor SC-1-181 to investigate the effects of dual inhibition. Our results indicated that dual inhibition of PI3K/Akt and MEK5/ERK5 signaling was more effective at reducing the proliferation and survival of TNBCs than single inhibition of either pathway alone. In particular, a loss of Bad phosphorylation at two distinct sites was observed with dual inhibition. Furthermore, the inhibition of both pathways led to p21 restoration, decreased cell proliferation, and induced apoptosis. In addition, the dual inhibition strategy was determined to be synergistic in MDA-MB-231 and BT-549 cells and was relatively nontoxic in the nonneoplastic MCF-10 cell line. In summary, the results from this study provide a unique prospective into the utility of a novel dual inhibition strategy for targeting TNBCs.

Topics: Apoptosis; Benzodiazepinones; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Survival; Drug Synergism; Female; Humans; MAP Kinase Kinase 5; Mitogen-Activated Protein Kinase 7; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Piperazines; Proto-Oncogene Proteins c-akt; Pyridones; Pyrimidines; Pyrimidinones; Signal Transduction; Triple Negative Breast Neoplasms

Triple-negative breast cancer (TNBC) is a very aggressive form of breast cancer with few molecularly targeted therapies. We used a novel unbiased approach to identify higher-order synergistic or enhancer combinations of marketed kinase inhibitor drugs that inhibit cell viability of TNBC cell lines. We mixed all 33 kinase-targeted drugs on the market at the time of this study, which allowed for all possible combinations to exist in the initial mixture. A kinase inhibitor group dropout approach was used to identify active groups and then single active drugs. After only three rounds of deconvolution, we identified five single drugs to test further. After further testing, we focused on one novel subset consisting of three kinase inhibitor drugs: dasatinib, afatinib, and trametinib (DAT) that target src family kinases, HER2/EGFR, and MEK, respectively. The DAT combination potently inhibited the proliferation of three TNBC cell lines and modestly inhibited a fourth. However, it was not significantly more potent or synergistic than other two drug combinations of these drugs. The cytotoxic activities of all possible combinations of these three drugs were also analyzed. Compared with all two-way combinations, the three-way DAT combination generated the most cytotoxicity and the highest synergies for two of the four cell lines tested, with possibly mild synergy in a third cell line. These data indicated that the DAT combination should be evaluated for efficacy in an in vivo model of TNBC and may provide a novel combination of existing drugs for the treatment of a subset of TNBC cases.

Topics: Afatinib; Animals; Cell Proliferation; Dasatinib; Drug Combinations; ErbB Receptors; Female; Humans; Mice; Mitogen-Activated Protein Kinase Kinases; Molecular Targeted Therapy; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; Receptor, ErbB-2; src-Family Kinases; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Due to its regulation of CDK1/2 phosphorylation, WEE1 plays essentially roles in the regulations of G2/M checkpoint and DNA damage response (DDR). WEE1 inhibition can increase genomic instability by inducing replication stress and G2/M checkpoint inactivation, which result in increased cellular sensitivity to DNA damaging agents. We considered an increase in genomic instability induced by WEE1 inhibition might be used to augment the effects of drugs targeting DNA repair protein. Typically, PARP inhibitors are effective in germline BRCA 1/2 mutated breast and ovarian cancer, but their applicabilities in triple-negative breast cancer (TNBC) are limited. This study was conducted to investigate the anti-tumor effects of the WEE1 inhibitor, AZD1775, and the mechanism responsible for its potentiation of sensitivity to olaparib (a PARP inhibitor) via the modulation of DDR in TNBC cells. Our results suggest that AZD1775 could be used to broaden the application range of olaparib in TNBC and provide a rationale for a clinical trial of combined olaparib and AZD1775 therapy.

Topics: Animals; Apoptosis; Cell Cycle; Cell Cycle Proteins; Cell Proliferation; DNA Damage; DNA Repair; Drug Resistance, Neoplasm; Drug Synergism; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Phthalazines; Piperazines; Poly(ADP-ribose) Polymerase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidinones; Triple Negative Breast Neoplasms; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Drug resistance in breast cancer cell populations has been shown to arise through phenotypic transition of cancer cells to a drug-tolerant state, for example through epithelial-to-mesenchymal transition or transition to a cancer stem cell state. However, many breast tumors are a heterogeneous mixture of cell types with numerous epigenetic states in addition to stem-like and mesenchymal phenotypes, and the dynamic behavior of this heterogeneous mixture in response to drug treatment is not well-understood. Recently, we showed that plasticity between differentiation states, as identified with intracellular markers such as cytokeratins, is linked to resistance to specific targeted therapeutics. Understanding the dynamics of differentiation-state transitions in this context could facilitate the development of more effective treatments for cancers that exhibit phenotypic heterogeneity and plasticity. In this work, we develop computational models of a drug-treated, phenotypically heterogeneous triple-negative breast cancer (TNBC) cell line to elucidate the feasibility of differentiation-state transition as a mechanism for therapeutic escape in this tumor subtype. Specifically, we use modeling to predict the changes in differentiation-state transitions that underlie specific therapy-induced changes in differentiation-state marker expression that we recently observed in the HCC1143 cell line. We report several statistically significant therapy-induced changes in transition rates between basal, luminal, mesenchymal, and non-basal/non-luminal/non-mesenchymal differentiation states in HCC1143 cell populations. Moreover, we validate model predictions on cell division and cell death empirically, and we test our models on an independent data set. Overall, we demonstrate that changes in differentiation-state transition rates induced by targeted therapy can provoke distinct differentiation-state aggregations of drug-resistant cells, which may be fundamental to the design of improved therapeutic regimens for cancers with phenotypic heterogeneity.

Topics: Antineoplastic Agents; Biomarkers, Tumor; Cell Death; Cell Differentiation; Cell Division; Cell Line, Tumor; Dimethyl Sulfoxide; Epithelial-Mesenchymal Transition; Female; Humans; Imidazoles; Models, Biological; Pyridones; Pyrimidinones; Quinolines; Triple Negative Breast Neoplasms

Triple negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that poses a clinical challenge. Thus, new therapy strategies are urgently needed. The selective WEE1 inhibitor, AZD1775, has shown strong anti-proliferative effects on a variety of tumors. Here, we first demonstrate that inhibition of ATR by selective inhibitor AZD6738 can enhance AZD1775-caused growth inhibition in TNBC. Our results show that the enhanced cell death is attributed to repressed DNA damage repair and excessive replication stress, thereby causing increased DNA damage reflected by accumulation of the DNA double-strand-break marker γH2AX. On the other hand, combined treatment with AZD6738 and AZD1775 forces mitotic entry of cells with DNA damages by activating CDK1 activity, inducing severely aberrant mitosis and mitotic catastrophe, ultimately resulting in cell death. Dual inhibition of WEE1 and ATR also inactivated RAD51-mediated homologous recombination, which sensitized TNBC cells to cisplatin and PARP inhibitor. Here, based on the preclinical results that ATR inhibition synergizes with WEE1 inhibition in TNBC, we propose that this combination therapy alone, or in parallel with chemotherapy, represents an innovative and potent targeted therapy in TNBC.

Topics: Animals; Antineoplastic Agents; Apoptosis; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Cell Death; Cell Line, Tumor; Cell Proliferation; DNA Damage; DNA Repair; Female; Humans; Indoles; MCF-7 Cells; Mice; Mitosis; Morpholines; Nuclear Proteins; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyrimidines; Pyrimidinones; Sulfonamides; Sulfoxides; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Poor prognosis in triple-negative breast cancer (TNBC) is due to an aggressive phenotype and lack of biomarker-driven targeted therapies. Overexpression of cyclin E and phosphorylated-CDK2 are correlated with poor survival in patients with TNBC, and the absence of CDK2 desensitizes cells to inhibition of Wee1 kinase, a key cell-cycle regulator. We hypothesize that cyclin E expression can predict response to therapies, which include the Wee1 kinase inhibitor, AZD1775.. Mono- and combination therapies with AZD1775 were evaluated in TNBC cell lines and multiple patient-derived xenograft (PDX) models with different cyclin E expression profiles. The mechanism(s) of cyclin E-mediated replicative stress were investigated following cyclin E induction or CRISPR/Cas9 knockout by a number of assays in multiple cell lines.. Cyclin E overexpression (i) is enriched in TNBCs with high recurrence rates, (ii) sensitizes TNBC cell lines and PDX models to AZD1775, (iii) leads to CDK2-dependent activation of DNA replication stress pathways, and (iv) increases Wee1 kinase activity. Moreover, treatment of cells with either CDK2 inhibitors or carboplatin leads to transient transcriptional induction of cyclin E (in cyclin E-low tumors) and result in DNA replicative stress. Such drug-mediated cyclin E induction in TNBC cells and PDX models sensitizes them to AZD1775 in a sequential treatment combination strategy.

Topics: Animals; Apoptosis; Bridged Bicyclo Compounds, Heterocyclic; Cell Cycle Proteins; Cell Line, Tumor; Cyclic N-Oxides; Cyclin E; Disease Models, Animal; DNA Repair; DNA Replication; Drug Resistance, Neoplasm; Gene Expression; Humans; Indolizines; Mice; Mice, Knockout; Models, Biological; Nuclear Proteins; Prognosis; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pyrazoles; Pyridinium Compounds; Pyrimidinones; Stress, Physiological; Triple Negative Breast Neoplasms; Xenograft Model Antitumor Assays

Hyper-activation of the MAPK and PI3K-AKT pathways is linked to tumour progression in triple-negative breast cancer (TNBC). However, clinically effective predictive markers for drugs targeted against protein kinases involved in these pathways have not been identified. We investigated the ability of MEK and PI3K catalytic activity to predict sensitivity to trametinib and wortmannin in TNBC. MEK and PI3K activities correlated strongly with each other only in cell lines showing wortmannin-specific sensitivity, as shown by a linear regression curve (R = 0.951). Accordingly, we created a new parameter that distinguishes trametinib and wortmannin sensitivity in vitro and in vivo. Our findings suggest that the catalytic activities of MEK and PI3K might predict the response of TNBC to trametinib and wortmannin.

Topics: Androstadienes; Animals; Antineoplastic Agents; Biocatalysis; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Female; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Mitogen-Activated Protein Kinase Kinases; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Pyridones; Pyrimidinones; Structure-Activity Relationship; Triple Negative Breast Neoplasms; Tumor Cells, Cultured; Wortmannin

The presence of tumor-infiltrating lymphocytes in triple-negative breast cancers is correlated with improved outcomes. Ras/MAPK pathway activation is associated with significantly lower levels of tumor-infiltrating lymphocytes in triple-negative breast cancers and while MEK inhibition can promote recruitment of tumor-infiltrating lymphocytes to the tumor, here we show that MEK inhibition adversely affects early onset T-cell effector function. We show that α-4-1BB and α-OX-40 T-cell agonist antibodies can rescue the adverse effects of MEK inhibition on T cells in both mouse and human T cells, which results in augmented anti-tumor effects in vivo. This effect is dependent upon increased downstream p38/JNK pathway activation. Taken together, our data suggest that although Ras/MAPK pathway inhibition can increase tumor immunogenicity, the negative impact on T-cell activity is functionally important. This undesirable impact is effectively prevented by combination with T-cell immune agonist immunotherapies resulting in superior therapeutic efficacy.MEK inhibition in breast cancer is associated with increased tumour infiltrating lymphocytes (TILs), however, MAPK activity is required for T cells function. Here the authors show that TILs activity following MEK inhibition can be enhanced by agonist immunotherapy resulting in synergic therapeutic effects.

Topics: 4-1BB Ligand; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Immunotherapy; Lymphocytes, Tumor-Infiltrating; Mammary Neoplasms, Animal; MAP Kinase Kinase 1; MAP Kinase Kinase 2; MAP Kinase Signaling System; Mice; OX40 Ligand; Protein Kinase Inhibitors; Pyridones; Pyrimidinones; T-Lymphocyte Subsets; T-Lymphocytes; Triple Negative Breast Neoplasms

Triple negative breast cancer (TNBC) is one of the most common invasive malignancies among women, associated with poor prognosis. Standard chemotherapy targets all dividing cells, resulting in dose-limiting toxicities. In this study, we demonstrated a strategy of encapsulating a hydrophobic synthetic compound, nifetepimine, having anticancer properties, in poly (lactic-co-glycolic acid) nanoparticles to increase selectivity of drug to cancerous cells with minimum toxicity towards normal cells. Nanoencapsulated nifetepimine (30-100nm) having loading and encapsulation efficiency of 7.45% and 75% respectively, was successfully internalized inside TNBC cells upon sustained release resulting in apoptosis. An in vivo bio-distribution study indicated that nanonifetepimine selectively accumulated into breast tumor sites of mice, primarily due to prolonged blood circulation time and binding of nifetepimine to epidermal growth factor receptor that remains overexpressed in most of the TNBC tumors. Moreover, we observed significant reduction in breast tumor volume with improved survival implying high tumor targetability of nanonifetepimine.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast; Cell Line, Tumor; Female; Humans; Mice; Nanoparticles; Pyrimidinones; Tissue Distribution; Triple Negative Breast Neoplasms

Triple negative breast cancers (TNBC) are among the most aggressive and therapy-resistant breast tumors and currently possess almost no molecular targets for therapeutic options in this horizon. In the present study we discerned the molecular mechanisms of potential interaction between the endoplasmic reticulum (ER) stress response and the MEK/ERK pathway in inducing apoptosis in TNBC cells. Here we observed that induction of ER stress alone was not sufficient to trigger significant apoptosis but simultaneous inhibition of the MEK/ERK pathway enhanced ER stress-induced apoptosis via a caspase-dependent mechanism. Our study also demonstrated nifetepimine, a dihydropyrimidone derivative as a potent anti-cancer agent in TNBC cells. Nifetepimine down-regulated the MEK/ERK pathway in MDAMB-231 and MDAMB-468 cells and resulted in blockage of ER stress-mediated GRP78 up-regulation. Detailed mechanistic studies also revealed that nifetepimine by down-regulating pERK expression also declined the promoter binding activity of TFII-I to the GRP78 promoter and in turn regulated GRP78 transcription. Studies further extended to in vivo Swiss albino and SCID mice models also revalidated the anti-carcinogenic property of nifetepimine. Thus our findings cumulatively suggest that nifetepimine couples two distinct signaling pathways to induce the apoptotic death cascade in TNBC cells and raises the possibility for the use of nifetepimine as a potent anti-cancer agent with strong immune-restoring properties for therapeutic intervention for this group of cancer bearers.

Topics: Animals; Apoptosis; Blotting, Western; Cell Cycle; Cell Proliferation; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Female; Heat-Shock Proteins; Humans; Immunoenzyme Techniques; Male; MAP Kinase Kinase Kinases; Mice; Mice, Nude; Mice, SCID; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Pyrimidinones; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Triple Negative Breast Neoplasms; Tumor Cells, Cultured; Xenograft Model Antitumor Assays