Single peptides and combination modalities for triple negative breast cancer

Unlike other types of breast cancers (BCs), no specific therapeutic targets have been established for triple negative breast cancer (TNBC). Therefore, chemotherapy and radiotherapy are the only available adjuvant therapeutic choices for TNBC. New emerging reports show that TNBC is associated with higher numbers of intratumoral tumor infiltrating lymphocytes. This is indicative of host anti-TNBC immune surveillance and suggesting that immunotherapy can be considered as a therapeutic approach for TNBC management. Recent progress in molecular mechanisms of tumor-immune system interaction and cancer vaccine development studies, fast discoveries and FDA approvals of immune checkpoint inhibitors, chimeric antigen receptor T-cells, and oncolytic virotherapy have significantly attracted attention and research directions toward the immunotherapeutic approach to TNBC. Here in this review different aspects of TNBC immunotherapies including the host immune system-tumor interactions, the tumor microenvironment, the relevant molecular targets for immunotherapy, and clinical trials in the field are discussed.     

Leisure-time physical activity is associated with reduced risks of breast cancer and triple negative breast cancer in Nigerian women

BackgroundLeisure-time physical activity(LTPA) is associated with a reduced risk of breast cancer, but this has less been investigated by cancer subtypes in Africans living in Sub-Saharan Africa(SSA). We examined the associations between LTPA and breast cancer including its subtypes in Nigerian women and explored the effect modification of body size on such associations.MethodsThe sample included 508 newly diagnosed primary invasive breast cancer cases and 892 controls from the Nigerian Integrative Epidemiology of Breast Cancer(NIBBLE) Study. Immunohistochemical(IHC) analysis was available for 294 cases. Total metabolic equivalents(METs) per hour/week of LTPA were calculated and divided by quartiles(Q1 <3.75, Q2:3.75–6.69, Q3:6.70–14.74, Q4:14.75 ≤). We applied logistic regressions to estimate the adjusted Odds Ratios(ORs) between LTPA and breast cancer and by its molecular subtypes and whether age-adjusted associations are modified by BMI.ResultsThe mean age(Mean±SD) of cases vs. controls(45.5 ± 11.1vs.40.1 ± 9.0) was higher, and the mean total METs hour/week was higher in controls vs. cases(11.9 ± 14.9vs.8.3 ± 11.1,p-value<0.001). Overall, 43.2%(N = 127/294) were classified as HRP, and 41.8%(N = 123/294) as TNBC. Women in the higher LTPA quartiles(Q3-Q4) vs. Q1 had lower odds of having breast cancer(OR_Q4vs.Q1=0.51,95%CI:0.35–0.74) and TNBC(OR_Q4vs.Q1=0.51, 95%CI:0.27–0.96), but not HRP(OR_Q4vs.Q1=0.61,95%CI:0.34–1.09) after adjusting for age, age at first menarche, body size, breastfeeding, menopausal, parity, contraceptives, demographics, alcohol, smoking, and physical activity at home and work. Lastly, LTPA and its age-adjusted association with breast cancer was more pronounced in women with BMI< 30 vs. BMI 30 + .ConclusionsLTPA may reduce the risk of breast cancer, especially TNBC, which is the more aggressive and prevalent molecular subtype of breast cancer in SSA.     

Caloric restriction coupled with radiation decreases metastatic burden in triple negative breast cancer

Purpose: Metastatic breast cancer is devastating and triple negative breast cancers (TNBC) have a higher propensity for metastasis. Improved local control upfront in this aggressive cancer could potentially decrease its propensity toward metastasis. We sought to determine if using caloric restriction (CR) as a systemic therapy, combined with radiation therapy (IR) to the primary tumor, may impact metastatic disease. Methods: An orthotopic mouse model using a highly metastatic, luciferase-tagged TNBC cell line (4T1), was used to generate palpable tumors. Mice were then treated with CR, IR, and a combination of the two. In vivo imaging was performed for metastatic evaluation. Molecular evaluation of the tumors was performed, generating a mechanistic hypothesis for CR, which was then tested with pertinent pathway inhibition in the model. Results: CR significantly increased the time to developing metastases, decreased the overall number and volume of lung metastases, and increased survival. CR decreased proliferation, increased apoptosis and globally downregulated the IGF-1R signaling pathway. Adding an IGF-1R/INSR inhibitor to local IR in vivo accomplished a decrease in metastases similar to CR plus IR, demonstrating the importance of the IGF-1R signaling pathway, and underscoring it as a possible mechanism for CR. Conclusions: CR decreased metastatic burden and therefore may complement cytotoxic therapies being used in the clinical setting for metastatic disease. Downregulation of the IGF-1R pathway, is in part responsible for this response and modulating IGF-1R directly resulted in similar improved progression-free survival. The novel use of CR has the potential to enhance clinical outcomes for patients with metastatic breast cancer.     

Enhanced cytotoxic activity of doxorubicin through the inhibition of autophagy in triple negative breast cancer cell line

BackgroundThe outcome of triple negative breast cancer is still poor and requires improvement with better therapy options. Autophagy has recently been shown to play a role in anticancer drug resistance. Therefore, we investigated if the effectiveness of doxorubicin was augmented by the inhibition of autophagy.MethodsMDA-MB-231 was used as a model cell line for triple negative breast cancer and 3-methyladenine was used as an inhibitor of autophagy. Cells were treated with 0.46–1.84 μM doxorubicin and 2.5–10 μM 3-methyladenine for 48 h. Cell death mode was examined with M30 and M65 ELISA assays. ROS level and LDH activity was examined and the cellular acidic compartment of cells was monitored by acridine orange staining. The expression of various autophagy and apoptosis related proteins/genes were evaluated with Western blotting and RT-qPCR respectively.ResultsSynergism was observed between the compounds (CI value < 1.0). RT-qPCR analysis revealed that the combination resulted in a down-regulation of autophagy-related genes. Moreover, the combination resulted in a different cell death modality, upregulating necroptosis-related genes. This suggests that the mode of cell death may switch from apoptosis to necroptosis, which is a more severe form of cell death, when autophagy is inhibited. These results were further confirmed at protein level by Western blotting.ConclusionInhibition of autophagy seems to sensitize triple negative breast cancer cells to doxorubicin, warranting further in vivo studies for the proof of this concept.General significanceAutophagy has a key role in drug resistance in MDA-MB-231 cells. Therefore combinatorial approaches may effectively overcome resistance.     

Antitumor activity of efrapeptins,alone or in combination with 2-deoxyglucose,in breast cancer in vitro and in vivo

Efrapeptins (EF), a family of fungal peptides, inhibit proteasomal enzymatic activities and the in vitro and in vivo growth of HT-29 cells. They are also known inhibitors of F₁F₀-ATPase, a mitochondrial enzyme that functions as an Hsp90 co-chaperone. We have previously shown that treatment of cancer cells with EF results in disruption of the Hsp90:F₁F₀-ATPase complex and inhibition of Hsp90 chaperone activity. The present study examines the effect of EF on breast cancer growth in vitro and in vivo. As a monotherapy, EF inhibited cell proliferation in vitro with an IC₅₀ value ranging from 6 nM to 3.4 μM. Inhibition of Hsp90 chaperone function appeared to be the dominant mechanism of action and the factor determining cellular sensitivity to EF. In vitro inhibition of proteasome became prominent in the absence of adequate levels of Hsp90 and F₁F₀-ATPase as in the case of the relatively EF-resistant MDA-MB-231 cell line. In vivo, EF inhibited MCF-7 and MDA-MB-231 xenograft growth with a maximal inhibition of 60% after administration of 0.15 and 0.3 mg/kg EF, respectively. 2-Deoxyglucose (2DG), a known inhibitor of glycolysis, acted synergistically with EF in vitro and antagonistically in vivo. In vitro, the synergistic effect was attributed to a prolonged endoplasmic reticulum (ER) stress. In vivo, the antagonistic effect was ascribed to the downregulation of tumoral and/or stromal F₁F₀-ATPase by 2DG.     

Diagnosis of triple negative breast cancer using expression data with several machine learning tools

Breast cancer is one of the top three commonly caused cancers worldwide. Triple Negative Breast Cancer (TNBC), a subtype of breast cancer, lacks expression of the oestrogen receptor, progesterone receptor, and HER2. This makes the prognosis poor and early detection hard. Therefore, AI based neural models such as Binary Logistic Regression, Multi-Layer Perceptron and Radial Basis Functions were used for differential diagnosis of normal samples and TNBC samples collected from signal intensity data of microarray experiment. Genes that were significantly upregulated in TNBC were compared with healthy controls. The MLP model classified TNBC and normal cells with anaccuracy of 93.4%. However, RBF gave 74% accuracy and binary Logistic Regression model showed an accuracy of 90.0% in identifying TNBC cases.     

Glabratephrin通过抑制P-糖蛋白逆转三阴性乳腺癌的阿霉素耐药

三阴性乳腺癌是最具侵略性的乳腺癌之一。目前首要的治疗方案是化疗,通常以蒽环类药物如阿霉素为主。然而,由于P-糖蛋白(Pgp)的存在,化疗的疗效受到限制。Pgp是一种膜转运蛋白,可以排出阿霉素,减少其细胞积累和疗效。因此,如何开发安全有效的药物抑制Pgp的活性是一个巨大的挑战。来自意大利都灵大学的Chiara Riganti及其团队证明了一种来自灰毛豆的异戊二烯基黄酮Glabratephrin(Glab)能增加阿霉素在高Pgp含量的三阴性乳腺癌细胞中的积累和细胞毒性。     

Featured Article: Teriflunomide,an immunomodulatory drug,exerts anticancer activity in triple negative breast cancer cells

Triple-negative breast cancer (TNBC) is defined as a group of primary breast cancers lacking expression of estrogen, progesterone, and human epidermal growth factor receptor-2 (HER-2) receptors, characterized by higher relapse rate and lower survival compared with other subtypes. Due to lack of identified targets and molecular heterogeneity, conventional chemotherapy is the only available option for treatment of TNBC, but non-discordant positive therapeutic efficacy could not be achieved. Here, we demonstrated that these TNBC cells were sensitive to teriflunomide, which was a well-known immunomodulatory drug for treatment of relapsing multiple sclerosis (MS). Potent anti-cancer effects in TNBC in vitro, including proliferation inhibition, cell cycle delay, cell apoptosis, and suppression of cell motility and invasiveness, could be achieved with this agent. Of note, we showed that multiple signals involved in TNBC proliferation, survival, migratory, and invasive potential were under regulation by teriflunomide. Among them, we identified down-regulation of growth factor receptors to abolish growth maintenance, suppression of c-Myc, and cyclin D1 to contribute to its anti-proliferative effect, modulation of components of cell cycle to induce S-phase arrest, degradation of Bcl-xL, and up-regulation of BAX via activation of MAPK pathway to induce apoptosis, and inhibition of epithelial-mesenchymal transition (EMT) process, matrix metalloproteinase-9 (MMP9) expression, and inactivation of Src/FAK to reduce TNBC migration and invasion. The results identified teriflunomide may be of therapeutic benefit for the more aggressive and difficult-to-treat breast cancer subtype, indicating the use of teriflunomide for clinical trials for treatment of TNBC patients.     

Thioridazine combined with carboplatin results in synergistic inhibition of triple negative breast cancer by targeting cancer stem cells

Cancer stem cells (CSCs) in triple-negative breast cancer (TNBC) are closely related to tumorigenesis and metastasis. Thioridazine (THZ) is a usual phenothiazine antipsychotic drug that can destroy CSCs. We aimed to explore whether THZ could sensitize metastatic TNBC cells, especially the CSCs, to carboplatin (CBP) treatment. Metastatic TNBC cells, 4T1 cells, and tumor-bearing mice were treated with THZ and CBP as monotherapy or combination therapy. MTT, flow cytometry, electron microscopy, immunohistochemistry and western blotting were applied to assess the cell viability, apoptosis, mitochondrial morphology and the relevant protein levels, respectively. Tumor size and lung metastasis under different treatments as well as tumorigenesis of residual tumor cells from each group were monitored. THZ combined with CBP inhibited 4T1 tumor cell proliferation and induced apoptosis by inhibiting the PI3K-AKT-mTOR pathway and activating estrogen receptor stress. THZ also showed strong activity against breast CSCs, THZ combined with CBP significantly destroyed cancer cells, inhibited lung metastasis and relieved the tumor burden; Our data demonstrated that THZ can sensitize TNBC cells to CBP treatment and this combination therapy may provide a bright strategy for TNBC treatment by targeting both cancer cells and CSCs.     

Antitumor activity of Herceptin in combination with STEALTH liposomal cisplatin or nonliposomal cisplatin in a HER2 positive human breast cancer model

Single agent antitumor activity of Herceptin, a humanized monoclonal antibody directed against HER2, has been demonstrated in numerous preclinical and clinical studies. Additionally, combination therapy with Herceptin and chemotherapy (CRx) has demonstrated additive antitumor activity in both preclinical models and early clinical trials. STEALTH (pegylated) liposomal (PL) cisplatin, also known as SPI-077, is currently in clinical trials for a variety of solid tumors. The three studies reported here discuss the antitumor activity of the combination of Herceptin and nonliposomal cisplatin or PL-cisplatin in two xenograft tumor models, initiated from the cell lines, BT474 and MDA453, that overexpress the oncogene, HER2. Herceptin alone had significant antitumor activity in all three experiments (p < 0.0001). Nonliposomal cisplatin and PL-cisplatin were both effective antitumor agents but, at tolerable dose levels, PL-cisplatin was superior to nonliposomal cisplatin (p < 0.0003). The effect of combining Herceptin with the chemotherapeutic cisplatin or PL-cisplatin, was most significant at moderate doses of H (0.5 mg/kg, p < 0.0001), but tended to be greater than either agent alone in all experiments. The combination of PL-cisplatin with Herceptin had statistically similar antitumor activity to that of nonliposomal cisplatin with Herceptin in all experiments. We conclude that combination therapy with PL-cisplatin and Herceptin results in significant antitumor activity with the potential for reducing toxicity in metastatic breast cancer patients.     

Metformin induces unique biological and molecular responses in triple negative breast cancer cells

Triple negative (TN) breast cancer is more frequent in women who are obese or have type II diabetes, as well as young Women of Color. These cancers do not express receptors for the steroid hormones estrogen or progesterone, or the type II receptor tyrosine kinase (RTK) Her-2 but do have upregulation of basal cytokeratins and the epidermal growth factor (EGFR). These data suggest that aberrations of glucose and fatty acid metabolism, signaling through EGFR and genetic factors may promote the development of TN cancers. The anti-type II diabetes drug metformin has been associated with a decreased incidence of breast cancer, although the specific molecular subtypes that may be reduced by metformin have not been reported. Our data indicates that metformin has unique anti-TN breast cancer effects both in vitro and in vivo. It inhibits cell proliferation (with partial S phase arrest), colony formation and induces apoptosis via activation of the intrinsic and extrinsic signaling pathways only in TN breast cancer cell lines. At the molecular level, metformin increases P-AMPK, reduces P-EGFR, EGFR, P-MAPK, P-Src, cyclin D1 and cyclin E (but not cyclin A or B, p27 or p21), and induces PARP cleavage in a dose- and time-dependent manner. These data are in stark contrast to our previously published biological and molecular effects of metformin on luminal A and B, or Her-2 type breast cancer cells. Nude mice bearing tumor xenografts of the TN line MDA-MB-231, treated with metformin, show significant reductions in tumor growth (p=0.0066) and cell proliferation (p=0.0021) as compared to untreated controls. Metformin pre-treatment, before injection of MDA-MB-231 cells, results in a significant decrease in tumor outgrowth and latency. Given the unique anti-cancer activity of metformin against TN disease, both in vitro and in vivo, it should be explored as a therapeutic agent against this aggressive form of breast cancer.     

RB loss contributes to aggressive tumor phenotypes in MYC-driven triple negative breast cancer

Triple negative breast cancer (TNBC) is characterized by multiple genetic events occurring in concert to drive pathogenic features of the disease. Here we interrogated the coordinate impact of p53, RB, and MYC in a genetic model of TNBC, in parallel with the analysis of clinical specimens. Primary mouse mammary epithelial cells (mMEC) with defined genetic features were used to delineate the combined action of RB and/or p53 in the genesis of TNBC. In this context, the deletion of either RB or p53 alone and in combination increased the proliferation of mMEC; however, the cells did not have the capacity to invade in matrigel. Gene expression profiling revealed that loss of each tumor suppressor has effects related to proliferation, but RB loss in particular leads to alterations in gene expression associated with the epithelial-to-mesenchymal transition. The overexpression of MYC in combination with p53 loss or combined RB/p53 loss drove rapid cell growth. While the effects of MYC overexpression had a dominant impact on gene expression, loss of RB further enhanced the deregulation of a gene expression signature associated with invasion. Specific RB loss lead to enhanced invasion in boyden chambers assays and gave rise to tumors with minimal epithelial characteristics relative to RB-proficient models. Therapeutic screening revealed that RB-deficient cells were particularly resistant to agents targeting PI3K and MEK pathway. Consistent with the aggressive behavior of the preclinical models of MYC overexpression and RB loss, human TNBC tumors that express high levels of MYC and are devoid of RB have a particularly poor outcome. Together these results underscore the potency of tumor suppressor pathways in specifying the biology of breast cancer. Further, they demonstrate that MYC overexpression in concert with RB can promote a particularly aggressive form of TNBC.     

The application of aptamer 5TR1 in triple negative breast cancer target therapy

Chemotherapy is one of the standard strategies for treatment of breast cancer. Adriamycin (Dox) is a first‐line chemotherapy agent for breast cancer. However, the gastrointestinal reactions, myocardial toxicity and other side effects caused by Dox due to its un‐specific cytotoxicity limit the clinical treatment effect. To address this need, aptamer has been regarded as an ideal target molecular carrier. In the present study, we selected an aptamer 5TR1 that can specifically bind to the MUC1 protein which has been regarded as an important tumor biomarker, as well as a potential target in anticancer therapies. Dox was loaded on the modified 5TR1‐GC, which specifically targets breast cancer cell MDA‐MB‐231. Cell viability and apoptosis assays demonstrated that the 5TR1‐GC‐Dox exhibited target specificity of cytotoxicity in MDA‐MB‐231. Moreover, in vivo xenograft study also confirmed that 5TR1‐GC‐Dox had a more effective effect on tumor growth inhibition and induced the apoptosis of malignant tumor cells compared to Dox. We provided a novel experimental and theoretical basis for developing an aptamer targeted drug system, thus to promote the killing effect of drugs on breast cells and to reduce the damage to normal cells and tissues for breast cancer.     

Nanobiopolymer for direct targeting and inhibition of EGFR expression in triple negative breast cancer

Treatment options for triple negative breast cancer (TNBC) are generally limited to cytotoxic chemotherapy. Recently, anti-epidermal growth factor receptor (EGFR) therapy has been introduced for TNBC patients. We engineered a novel nanobioconjugate based on a poly(β-L-malic acid) (PMLA) nanoplatform for TNBC treatment. The nanobioconjugate carries anti-tumor nucleosome-specific monoclonal antibody (mAb) 2C5 to target breast cancer cells, anti-mouse transferrin receptor (TfR) antibody for drug delivery through the host endothelial system, and Morpholino antisense oligonucleotide (AON) to inhibit EGFR synthesis. The nanobioconjugates variants were: (1) P (BioPolymer) with AON, 2C5 and anti-TfR for tumor endothelial and cancer cell targeting, and EGFR suppression (P/AON/2C5/TfR), and (2) P with AON and 2C5 (P/AON/2C5). Controls included (3) P with 2C5 but without AON (P/2C5), (4) PBS, and (5) P with PEG and leucine ester (LOEt) for endosomal escape (P/mPEG/LOEt). Drugs were injected intravenously to MDA-MB-468 TNBC bearing mice. Tissue accumulation of injected nanobioconjugates labeled with Alexa Fluor 680 was examined by Xenogen IVIS 200 (live imaging) and confocal microscopy of tissue sections. Levels of EGFR, phosphorylated and total Akt in tumor samples were detected by western blotting. In vitro western blot showed that the leading nanobioconjugate P/AON/2C5/TfR inhibited EGFR synthesis significantly better than naked AON. In vivo imaging revealed that 2C5 increased drug-tumor accumulation. Significant tumor growth inhibition was observed in mice treated with the lead nanobioconjugate (1) [P = 0.03 vs. controls; P<0.05 vs. nanobioconjugate variant (2)]. Lead nanobioconjugate (1) also showed stronger inhibition of EGFR expression and Akt phosphorylation than other treatments. Treatment of TNBC with the new nanobioconjugate results in tumor growth arrest by inhibiting EGFR and its downstream signaling intermediate, phosphorylated Akt. The nanobioconjugate represents a new generation of nanodrugs for treatment of TNBC.     

LIN9 confers paclitaxel resistance in triple negative breast cancer cells by upregulating CCSAP

LIN9 functions to regulate cell mitotic process.Dysregulation of LIN9 expression is associated with development of human cancers.In this study we assessed the association of LIN9 expression with paclitaxel resistance and clarified the underlying mechanisms for the first time.LIN9 expression in breast cancer tissues was retrieved from publicly available online databases and statistically analyzed.Human TNBC cell lines MDA-MB-231 and MDA-MB-468 and their corresponding paclitaxelresistant sublines 231PTX and 468PTX were used to assess the expression of LIN9 by qRT-PCR and Western blot,cell growth by cell counting,cell viability by MTS assay,and cell apoptosis by flow cytometry.The data showed that high LIN9 expression in breast cancer patients receiving chemotherapy was related to poor overall survival (OS).LIN9 expression was upregulated in paclitaxel-resistant TNBC cells compared to their parental cells.Knockdown of LIN9 or treatment of paclitaxel-resistant TNBC cells with a bromo-and extra-terminal domain inhibitor (BETi) JQ1 which also decreased LIN9 expression enhanced the sensitivity of paclitaxel-resistant TNBC cells to paclitaxel.Mechanistically,decreased LIN9 in resistant cell lines reduced tumor cell viability,promoted multinucleated cells formation and induced tumor cell apoptosis,potentially by directly regulating microtubule-binding protein CCSAP.In conclusion,high LIN9 expression contributed to poor clinical outcomes and paclitaxel resistance in TNBC and BETi,targeting LIN9 expression,could be a reversible drug for PTX-resistant TNBC patients.