Triple Receptor-negative Breast Cancer Research Articles

Revealing the Potential Markers of N(4)-Acetylcytidine through acRIP-seq in Triple-Negative Breast Cancer.

Understanding the causes of tumorigenesis and progression in triple-receptor negative breast cancer (TNBC) can help the design of novel and personalized therapies and prognostic assessments. Abnormal RNA modification is a recently discovered process in TNBC development. TNBC samples from The Cancer Genome Atlas database were categorized according to the expression level of NAT10, which drives acetylation of cytidine in RNA to N(4)-acetylcytidine (ac4C) and affects mRNA stability. A total of 703 differentially expressed long non-coding RNAs (lncRNAs) were found between high- and low-expressed NAT10 groups in TNBC. Twenty of these lncRNAs were significantly associated with prognosis. Two breast cancer tissues and their paired normal tissues were sequenced at the whole genome level using acetylated RNA immunoprecipitation sequencing (acRIP-seq) technology to identify acetylation features in TNBC, and 180 genes were significantly differentially ac4c acetylated in patients. We also analyzed the genome-wide lncRNA expression profile and constructed a co-expression network, containing 116 ac4C genes and 1080 lncRNAs. Three of these lncRNAs were prognostic risk lncRNAs affected by NAT10 and contained in the network. The corresponding reciprocal pairs were "LINC01614-COL3A1", "OIP5-AS1-USP8", and "RP5-908M14.9-TRIR". These results indicate that RNA ac4c acetylation involves lncRNAs and affects the tumor process and prognosis of TNBC. This will aid the prediction of drug targets and drug sensitivity.

Matrix metalloproteinase 2 is a target of the RAN-GTP pathway and mediates migration, invasion and metastasis in human breast cancer

RAS-related nuclear protein(RAN) is a nuclear shuttle and normally regulates events in the cell cycle. When overexpressed in cultured cells, it causes increases in cell migration/invasion in vitro and its overexpression is associated with early breast cancer patient deaths in vivo. However, the underlying mechanism is unknown. The effect of RAN overexpression on potential targets MMP2, ATF3, CXCR3 was investigated by Real-Time PCR/Western blots in the triple receptor negative breast cancer(TRNBC) cell line MDA-MB231 and consequent biological effects were measured by cell adhesion, cell migration and cell invasion assays. Results showed that knockdown of RAN lead to a reduction of MMP2 and its potential regulators ATF3 and CXCR3. Moreover, knockdown of ATF3 or CXCR3 downregulated MMP2 without affecting RAN, indicating that RAN regulates MMP2 through ATF3 and CXCR3. Knockdown of RAN and MMP2 reduced cell adhesion, cell migration and cell growth in agar, whilst overexpression of MMP2 reversed the knockdown of RAN. Furthermore, immunohistochemical staining for RAN and MMP2 are positively associated with each other in the same tumour and separately with patient survival times in breast cancer specimens, suggesting that a high level of RAN may be a pre-requisite for MMP2 overexpression and metastasis. Moreover, positive immunohistochemical staining for both RAN and MMP-2 reduces further patient survival times over that for either protein separately. Our results suggest that MMP2 expression can stratify progression of breast cancers with a high and low incidence of RAN, both RAN and MMP2 in combination can be used for a more accurate patient prognosis. Simple summaryRan is an important regulator of normal cell growth and behaviour. We have established in cell line models of breast cancer (BC) a molecular pathway between RAN and its protein-degrading effector MMP-2 and properties related to metastasis in culture. Using immunohistochemistry (IHC) staining of primary BCs, we have shown that RAN and MMP-2 are on their own significantly associated with patient demise from metastatic BC. Moreover, when staining for MMP-2 is added to that for RAN in the primary tumours, there is a significant decrease in patient survival time over that for either protein alone. Thus a combination of staining for RAN and MMP2 is an excellent marker for poor prognosis in breast cancer.

Two year results of blessed: Expanded access for deltarex-g for an intermediate size population with advanced pancreatic cancer and sarcoma (NCT04091295) and individual use IND for EARLY-STAGE invasive carcinoma of breast (IND# 19130) .

e15048 Background: Defects in cell cycle control are fundamental oncogenic drivers and targeting deregulated cell cycling is under intensive study. Cell cycle cyclin G1 (CCNG1) inhibitor therapy, exemplified by DeltaRex-G, a tumor-targeted retro vector encoding a cytocidal CCNG1 inhibitor gene, has been tested in over 280 cancer patients worldwide in early studies, inducing long term (10-13 years) survival in certain patients with intractable metastatic pancreatic adenocarcinoma, sarcoma, and breast cancer. Hence, further clinical development of DeltaRex-G for cancer patients who have few or no therapeutic options is apropos. Methods: Primary objective: To determine overall survival. Secondary objective: To evaluate disease control, best overall response, and incidence of treatment-related adverse events. Patient and Methods: Study 1 is entitled “Blessed: Expanded Access for DeltaRex-G for Advanced Pancreatic Cancer and Sarcoma (NCT04091295)”. Study 2 is entitled “Compassionate Use of DeltaRex-G for Advanced Cancers. In both studies, patients will receive DeltaRex-G at 1-3 x 10e11 cfu i.v. over 15-30 minutes, 3 x a week until significant disease progression or unacceptable toxicity occurs. Results: Seventeen patients were enrolled, 9 sarcomas, 2 pancreatic adenocarcinomas, 1 non-small cell lung cancer, 2 breast carcinoma, 1 prostate cancer, 1 cholangiocarcinoma, and 1 basal cell carcinoma and actinic keratosis. Three patients were enrolled in Study 1 and 14 patients were enrolled in Study 2. Two patients were initially enrolled in Study 1 and later enrolled in Study 2. Twelve of 17 enrolled patients were treated with DeltaRex-G monotherapy or in combination with FDA-approved cancer therapies. Of the 12 treated patients, 5 are alive 10 to 30 months from DeltaRex-G treatment initiation. Two patients with early-stage triple receptor-positive and triple receptor-negative breast cancer who received DeltaRex-G as adjuvant/first-line therapy are alive one year in complete remission; 2 patients with chemo-resistant Stage 4 sarcoma are alive 2 1/2 years, and one patient with advanced basal cell carcinoma is alive 10 months from treatment initiation. There were no treatment-related adverse events reported. Conclusions: Taken together,the data suggest that (1) Adjuvant/first-line therapy with DeltaRex-G may reduce the incidence of recurrence of early-stage invasive carcinoma of the breast and (2) DeltaRex-G may evoke tumor growth stabilization after failing standard chemotherapy. Phase 2 studies are planned to evaluate if DeltaRex-G (1) will reduce the incidence of recurrence in early-stage invasive carcinoma of breast, (2) improve survival in pancreatic cancer, and (3) prolong progression-free survival and overall survival in advanced sarcoma. Clinical trial information: NCT04091295.

ZMIZ2 promotes the development of triple-receptor negative breast cancer

BackgroundTriple-receptor negative breast cancer (TNBC) is an aggressive breast tumor subtype that generally has a poor prognosis. This study aimed to investigate the role and regulatory mechanisms of Zinc finger MIZ-type containing 2 (ZMIZ2) in relation to TNBC.MethodsBased on data from The Cancer Genome Atlas (TCGA), the expression of ZMIZ2 in different subtypes and its correlation with androgen receptor (AR) were analyzed, and a regulatory mechanism network was constructed. The expression and prognostic value of ZMIZ2 in clinical TNBC tissue samples were also investigated. Furthermore, in vitro studies were conducted to investigate the effects of ZMIZ2 knockdown on the malignant behaviors of TNBC cells and target gene expression.ResultsBased on TCGA data, ZMIZ2 was found to be significantly upregulated in TNBC tissues and its expression was negatively correlated with AR expression. Key relationships, such as the ZMIZ2-CCL5, ZMIZ2/AR-MCM3, ZMIZ2/AR-E2F4, and the ZMIZ2/AR-DHX38 were identified, which were enriched in NOD-like receptor signaling pathway/toll-like receptor signaling pathway, DNA replication, cell cycle, and spliceosome, respectively. Moreover, ZMIZ2 was upregulated in clinical breast cancer tissues and its high expression was correlated with the poor prognosis of TNBC patients. Furthermore, ZMIZ2 expression was increased in breast cancer cells, and a knockdown of ZMIZ2 inhibited MDA-MB-231 cell proliferation, migration, and invasion, induced cell cycle arrest in the G1 phase, and promoted cell apoptosis. Furthermore, ZMIZ2 knockdown inhibited the mRNA and protein expression of CCL5, MCM3, E2F4, and DHX38.ConclusionOur findings reveal that ZMIZ2 is upregulated in TNBC tissues and is associated with its poor prognosis. ZMIZ2 may promote TNBC progression by promoting the expression of its target genes and affecting the corresponding pathways. Consequently, ZMIZ2 may serve as a promising target for future TNBC treatments.

Investigations on Zinc Isotope Fractionation in Breast Cancer Tissue Using in vitro Cell Culture Uptake-Efflux Experiments.

Zinc (Zn) accumulates in breast cancer tumors compared to adjacent healthy tissue. Clinical samples of breast cancer tissue show light Zn isotopic compositions (δ66Zn) relative to healthy tissue. The underlying mechanisms causing such effects are unknown. To investigate if the isotopic discrimination observed for in vivo breast cancer tissue samples can be reproduced in vitro, we report isotopic data for Zn uptake-efflux experiments using a human breast cancer cell line. MDA-MB-231 cell line was used as a model for triple receptor negative breast cancer. We determined Zn isotope fractionation for Zn cell uptake (Δ66Znuptake) and cell efflux (Δ66Znefflux) using a drip-flow reactor to enable comparison with the in vivo environment. The MDA-MB-231 cell line analyses show Zn isotopic fractionations in an opposite direction to those observed for in vivo breast cancer tissue. Uptake of isotopically heavy Zn (Δ66Znuptake = +0.23 ± 0.05‰) is consistent with transport via Zn transporters (ZIPs), which have histidine-rich binding sites. Zinc excreted during efflux is isotopically lighter than Zn taken up by the cells (Δ66Znefflux = −0.35 ± 0.06‰). The difference in Zn isotope fractionation observed between in vitro MDA-MB-231 cell line experiments and in vivo breast tissues might be due to differences in Zn transporter levels or intercellular Zn storage (endoplasmic reticulum and/or Zn specific vesicles); stromal cells, such as fibroblasts and immune cells. Although, additional experiments using other human breast cancer cell lines (e.g., MCF-7, BT-20) with varying Zn protein characteristics are required, the results highlight differences between in vitro and in vivo Zn isotope fractionation.

Comprehensive mutagenesis identifies the peptide repertoire of a p53 T-cell receptor mimic antibody that displays no toxicity in mice transgenic for human HLA-A*0201.

T-cell receptor mimic (TCRm) antibodies have expanded the repertoire of antigens targetable by monoclonal antibodies, to include peptides derived from intracellular proteins that are presented by major histocompatibility complex class I (MHC-I) molecules on the cell surface. We have previously used this approach to target p53, which represents a valuable target for cancer immunotherapy because of the high frequency of its deregulation by mutation or other mechanisms. The T1-116C TCRm antibody targets the wild type p5365-73 peptide (RMPEAAPPV) presented by HLA-A*0201 (HLA-A2) and exhibited in vivo efficacy against triple receptor negative breast cancer xenografts. Here we report a comprehensive mutational analysis of the p53 RMPEAAPPV peptide to assess the T1-116C epitope and its peptide specificity. Antibody binding absolutely required the N-terminal arginine residue, while amino acids in the center of the peptide contributed little to specificity. Data mining the immune epitope database with the T1-116C binding consensus and validation of peptide recognition using the T2 stabilization assay identified additional tumor antigens targeted by T1-116C, including WT1, gp100, Tyrosinase and NY-ESO-1. Most peptides recognized by T1-116C were conserved in mice and human HLA-A2 transgenic mice showed no toxicity when treated with T1-116C in vivo. We conclude that comprehensive validation of TCRm antibody target specificity is critical for assessing their safety profile.

Epigenetic Modulation of SPCA2 Reverses Epithelial to Mesenchymal Transition in Breast Cancer Cells.

Simple SummaryThe triple receptor negative breast cancer subtype, or TNBC, currently has no tailored treatment options. TNBC is highly metastatic, associated with high patient mortality, and disproportionately occurs in Black/African American women where it contributes to racial disparities in health outcomes. Therefore, we focused on new therapeutic approaches to TNBC. We discovered that levels of the Calcium-ATPase SPCA2 are abnormally low in TNBC and that these low levels correlate with poor survival prognosis in patients. Previously, we showed that recombinant SPCA2 prevented TNBC cells from acquiring aggressive “mesenchymal” properties associated with metastasis both in vitro and in vivo. These findings motivated us to search for drugs that turn the SPCA2 gene back on in TNBC cells. In this study, we show that histone deacetylase inhibitors increase SPCA2 levels, activate Ca2+ signaling and convert cancer cells to a less aggressive “epithelial” state. These findings could lead to new treatment options for TNBC.The secretory pathway Ca2+-ATPase SPCA2 is a tumor suppressor in triple receptor negative breast cancer (TNBC), a highly aggressive molecular subtype that lacks tailored treatment options. Low expression of SPCA2 in TNBC confers poor survival prognosis in patients. Previous work has established that re-introducing SPCA2 to TNBC cells restores basal Ca2+ signaling, represses mesenchymal gene expression, mitigates tumor migration in vitro and metastasis in vivo. In this study, we examined the effect of histone deacetylase inhibitors (HDACi) in TNBC cell lines. We show that the pan-HDACi vorinostat and the class I HDACi romidepsin induce dose-dependent upregulation of SPCA2 transcript with concurrent downregulation of mesenchymal markers and tumor cell migration characteristic of epithelial phenotype. Silencing SPCA2 abolished the ability of HDACi to reverse epithelial to mesenchymal transition (EMT). Independent of ATPase activity, SPCA2 elevated resting Ca2+ levels to activate downstream components of non-canonical Wnt/Ca2+ signaling. HDACi treatment led to SPCA2-dependent phosphorylation of CAMKII and β-catenin, turning Wnt signaling off. We conclude that SPCA2 mediates the efficacy of HDACi in reversing EMT in TNBC by a novel mode of non-canonical Wnt/Ca2+ signaling. Our findings provide incentive for screening epigenetic modulators that exploit Ca2+ signaling pathways to reverse EMT in breast tumors.

Increased expression of glutamine transporter SNAT2/SLC38A2 promotes glutamine dependence and oxidative stress resistance, and is associated with worse prognosis in triple-negative breast cancer

BackgroundGlutamine (Gln) is an abundant nutrient used by cancer cells. Breast cancers cells and particularly triple-receptor negative breast cancer (TNBC) are reported to be dependent on Gln to produce the energy required for survival and proliferation. Despite intense research on the role of the intracellular Gln pathway, few reports have focussed on Gln transporters in breast cancer and TNBC.MethodsThe role and localisation of the Gln transporter SLC38A2/SNAT2 in response to Gln deprivation or pharmacological stresses was examined in a panel of breast cancer cell lines. Subsequently, the effect of SLC38A2 knockdown in Gln-sensitive cell lines was analysed. The prognostic value of SLC38A2 in a cohort of breast cancer was determined by immunohistochemistry.ResultsSLC38A2 was identified as a strongly expressed amino acid transporter in six breast cancer cell lines. We confirmed an autophagic route of degradation for SLC38A2. SLC38A2 knockdown decreased Gln consumption, inhibited cell growth, induced autophagy and led to ROS production in a subgroup of Gln-sensitive cell lines. High expression of SLC38A2 protein was associated with poor breast cancer specific survival in a large cohort of patients (p = 0.004), particularly in TNBC (p = 0.02).ConclusionsThese results position SLC38A2 as a selective target for inhibiting growth of Gln-dependent breast cancer cell lines.

Abstract 3561: HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition

Abstract Increased expression of Hypoxia-Inducible Factor 1α (HIF-1α) in breast tumours is associated with poor survival outcome. HIF-1α promotes phenotypic adaptations including metabolic reorganisation, angiogenesis and increased cell invasiveness. Notably, HIF-1α activity is increased in Triple receptor Negative Breast Cancer (TNBC) compared with other breast cancer subtypes. One reason for this elevated HIF-1α stabilisation in TNBC has been described; intracellular cysteine depletion which leads to impaired prolyl hydroxylase-domain (PHD) enzyme activity. Normally, cystine is acquired in exchange for intracellular glutamate using the xCT antiporter. However, in TNBC the excessive secretion and extracellular accumulation of glutamate impairs cystine exchange. Since the majority of glutamate produced in TNBC is derived from glutaminase-catalysed hydrolysis of glutamine, we hypothesised that glutaminase inhibition using a potent and selective inhibitor, CB-839, would improve glutamate/cystine exchange and reduce HIF-1α levels. We detected normoxic HIF-1α expression in BT549, Hs578T and SUM159PT TNBC cell cultures. In these cases HIF-1α protein was destabilised upon treatment with CB-839 and the expression of some HIF-1α-target genes was significantly decreased (e.g. CA9, BNIP3, PDK1, LDHA) but not others (e.g. ADM and VEGFA). HIF-1α destabilisation corresponded with increased PHD-dependent hydroxylation, supporting the hypothesis that glutaminase inhibition can improve PHD function. Consistently, WT but not a proline hydroxylation-null mutant HIF-1α protein was depleted by CB-839. We confirmed that CB-839 decreased glutamate secretion but, unexpectedly, HIF-1α levels were not restabilised by addition of excess extracellular glutamate. Thus glutamine metabolism drives normoxic HIF-1α stabilisation via an additional mechanism distinct from perturbation of the glutamate/cystine exchange system. To elucidate the underlying process we focused on the glutamine-derived metabolite (S)-2-Hydroxyglutarate [(S)-2HG], a competitive inhibitor of 2-Oxoglutarate (2OG)-dependent enzymes, including the PHD enzymes. Glutaminase inhibition decreased the levels of (S)-2HG. Furthermore, growing CB-839-treated cells in culture medium containing cell permeable (S)-2HG, but not (R)-2HG, could restabilise HIF-1α. In addition, increasing 2OG levels in these cells further stabilised HIF-1α, an effect that was blunted by inhibition of lactate dehydrogenase A (LDHA), a promiscuous source of (S)-2HG generated via reduction of 2OG. Conclusion: CB-839 has demonstrated encouraging clinical responses in patients with heavily pretreated and therapy-resistant metastatic TNBC. Our work demonstrates that high levels of glutamine metabolism provoke HIF-1α stabilisation through the LDHA-catalysed production of (S)-2HG. Assessment of HIF-1α activity may be a useful approach to identify glutamine-addicted tumours and predict responsiveness to glutaminase inhibitor therapy. Citation Format: Laura Cussonneau, Anne-Lise Dechaume, Pamela M. Murray, Henegama Liyanage, Tet-Woo Lee, Frederik Pruijn, Euphemia Y. Leung, Dean C. Singleton. HIF-1α stabilisation in triple receptor negative breast cancer is sensitive to glutaminase inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3561.

Pyruvate anaplerosis is a mechanism of resistance to pharmacological glutaminase inhibition in triple-receptor negative breast cancer

BackgroundGlutamine serves as an important nutrient with many cancer types displaying glutamine dependence. Following cellular uptake glutamine is converted to glutamate in a reaction catalysed by mitochondrial glutaminase. This glutamate has many uses, including acting as an anaplerotic substrate (via alpha-ketoglutarate) to replenish TCA cycle intermediates. CB-839 is a potent, selective, orally bioavailable inhibitor of glutaminase that has activity in Triple receptor-Negative Breast Cancer (TNBC) cell lines and evidence of efficacy in advanced TNBC patients.MethodsA panel of eleven breast cancer cell lines was used to investigate the anti-proliferative effects of the glutaminase inhibitors CB-839 and BPTES in different types of culture medium, with or without additional pyruvate supplementation. The abundance of the TCA cycle intermediate fumarate was quantified as a measure if TCA cycle anaplerosis. Pyruvate secretion by TNBC cultures was then assessed with or without AZD3965, a monocarboxylate transporter 1 (MCT1) inhibitor. Finally, two dimensional (2D) monolayer and three dimensional (3D) spheroid assays were used to compare the effect of microenvironmental growth conditions on CB-839 activity.ResultsThe anti-proliferative activity of CB-839 in a panel of breast cancer cell lines was similar to published reports, but with a major caveat; growth inhibition by CB-839 was strongly attenuated in culture medium containing pyruvate. This pyruvate-dependent attenuation was also observed with a related glutaminase inhibitor, BPTES. Studies demonstrated that exogenous pyruvate acted as an anaplerotic substrate preventing the decrease of fumarate in CB-839-treated conditions. Furthermore, endogenously produced pyruvate secreted by TNBC cell lines was able to act in a paracrine manner to significantly decrease the sensitivity of recipient cells to glutaminase inhibition. Suppression of pyruvate secretion using the MCT1 inhibitor AZD3965, antagonised this paracrine effect and increased CB-839 activity. Finally, CB-839 activity was significantly compromised in 3D compared with 2D TNBC culture models, suggesting that 3D microenvironmental features impair glutaminase inhibitor responsiveness.ConclusionThis study highlights the potential influence that both circulating and tumour-derived pyruvate can have on glutaminase inhibitor efficacy. Furthermore, it highlights the benefits of 3D spheroid cultures to model the features of the tumour microenvironment and improve the in vitro investigation of cancer metabolism-targeted therapeutics.

Randomized, phase II trial to evaluate the efficacy and safety of atezolizumab plus capecitabine adjuvant therapy compared to capecitabine monotherapy for triple receptor-negative breast cancer (TNBC) with residual invasive cancer after neoadjuvant chemotherapy (MIRINAE trial, KCSG-BR18-21).

TPS597 Background: Triple negative breast cancer (TNBC), lack of ER, PR and HER2 expression, is known to have aggressive clinical features such as early recurrence, drug resistance, and frequent distant metastasis at the diagnosis. The most effective chemotherapy combinations used for early TNBC include anthracycline, taxanes, and/or platinum agents. Achieving a pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC) provides important prognostic information and is considered as a surrogate endpoint in many clinical trials especially with TNBC. Patients with residual invasive disease after NAC have a high risk for early relapse and worse prognosis compared to those with pCR. Therefore, patients who did not get pCR could be better candidates for additional adjuvant treatment because their risk of recurrence would be higher than those with pCR. The CREATE-X (capecitabine for residual cancer as adjuvant therapy) trial howed that adjuvant capecitabine treatment improved 5-yr rate of disease free survival in TNBC subtype. A recent study indicated that immunosuppressive microenvironment had developed even in early stage of TNBC with increased T cells with a high exhaustion signature which are targets of immune modulating agents. Therefore, earlier cooperation of immune modulating drugs would be beneficial by generating a long-lasting anti-tumor immune response to micrometastatic disease, thus preventing disease relapse or recurrence. Methods: This study is a phase II, multicenter, randomized open label trial of atezolizumab (anti-PD-L1 antibody) and capecitabine compared with capecitabine in patients with TNBC who had residual disease after NAC. 284 patients will be enrolled from 15 sites in Korea with a primary objective to access the 5-yr invasive disease-free survival (IDFS) rate. Secondary objectives include 5-yr IDFS rate in PD-L1 positive population, distant relapse free survival (DRFS), overall survival (OS), and safety. Major inclusion and exclusion criteria are followings; 1) histologically confirmed TNBC, 2) received anthracycline and taxane based NAC followed by complete breast surgery, 3) residual disease after NAC must be ≥1cm in the greatest dimension, and/or have macroscopically positive lymph nodes. The study is open with 13 patients enrolled at the time of submission. Clinical trial information: NCT03756298 .

Development of Therapeutic Anti-JAGGED1 Antibodies for Cancer Therapy.

The role of Notch signaling and its ligand JAGGED1 (JAG1) in tumor biology has been firmly established, making them appealing therapeutic targets for cancer treatment. Here, we report the development and characterization of human/rat-specific JAG1-neutralizing mAbs. Epitope mapping identified their binding to the Notch receptor interaction site within the JAG1 Delta/Serrate/Lag2 domain, where E228D substitution prevented effective binding to the murine Jag1 ortholog. These antibodies were able to specifically inhibit JAG1-Notch binding in vitro, downregulate Notch signaling in cancer cells, and block the heterotypic JAG1-mediated Notch signaling between endothelial and vascular smooth muscle cells. Functionally, in vitro treatment impaired three-dimensional growth of breast cancer cell spheroids, in association with a reduction in cancer stem cell number. In vivo testing showed variable effects on human xenograft growth when only tumor-expressed JAG1 was targeted (mouse models) but a more robust effect when stromal-expressed Jag1 was also targeted (rat MDA-MB-231 xenograft model). Importantly, treatment of established triple receptor-negative breast cancer brain metastasis in rats showed a significant reduction in neoplastic growth. MRI imaging demonstrated that this was associated with a substantial improvement in blood-brain barrier function and tumor perfusion. Lastly, JAG1-targeting antibody treatment did not cause any detectable toxicity, further supporting its clinical potential for cancer therapy.

Activation of Lxrr Causes Metabolic Reprogramming and Sensitizes Solid Tumors to Bcl-xL Inhibition Mediated Apoptosis

Liver-X-receptor agonists (LXR) have been shown to bear anti-tumor activity. However, their efficacy is limited and therefore additional strategies to further enhance their anti-cancer activity are warranted. By uncovering novel and unexpected mechanisms through transcriptome and gene set enrichment analysis coupled with global untargeted metabolite screening, we show that LXR agonists, LXR623 and GW3965, enhance the anti-proliferative effects of clinically validated as well as novel selective BH3-mimetics in solid tumor malignancies, including glioblastoma, colon carcinoma, melanoma and triple-receptor negative breast cancer. LXR agonists and ABT263 acted in a synergistic fashion to reduce proliferation of cancer cells, which was predominantly mediated by enhanced apoptosis. Inhibition of Bcl-xL was most relevant for the combination treatment to exert its effects. Extracellular flux analysis revealed that activation of LXRβ resulted in reprogramming of tumor cell metabolism, leading to suppression of mitochondrial respiration, which occurred independent of cholesterol. Instead, LXR activation engages in a transcriptional program to suppress mitochondrial metabolism. In turn, this leads to a subsequent decline in ATP levels, culminating in endoplasmic reticulum stress with upregulation of pro-apoptotic Noxa in an ATF4 dependent manner. Consistently, siRNA mediated suppression of Noxa rescued from the combination treatment of BH3-mimetics and LXR agonists. In conventional and patient-derived xenograft models of colon carcinoma, melanoma and glioblastoma, the combination treatment of ABT263 and LXR agonists reduced tumor sizes significantly stronger than single treatments. Therefore, the combination treatment of LXR agonists and BH3-mimetics might be a viable efficacious treatment approach for solid malignancies.

Abstract OT1-02-04: SWOG S1418/NRG -BR006: A randomized, phase III trial to evaluate the efficacy and safety of MK-3475 as adjuvant therapy for triple receptor-negative breast cancer with > 1 cm residual invasive cancer or positive lymph nodes (>pN1mic) after neoadjuvant chemotherapy

Abstract Background: Patients with residual cancer after neoadjuvant chemotherapy, particularly triple negative cancers (TNBC), have poor prognosis.The SWOG S1418 / NRG BR-006 (NCT02954874) randomized, phase III trial tests the hypothesis that administration of pembrolizumab after surgery for 12 months will reduce invasive disease-free survival (IDFS) by 33% compared to observation in patients with TNBC and > 1 cm residual invasive cancer or positive lymph nodes (>pN1mic) after neoadjuvant chemotherapy. Methods: Eligible patients ≥18 years old with triple negative breast cancer defined by ASCO/CAP guidelines and >1 cm residual invasive cancer in the breast, or any macrometastases in the lymph nodes after completion of 16-24 weeks of neoadjuvant chemotherapy. Patients may receive post-operative chemotherapy for up to 24 weeks but must be registered for screening within 35 days of completion of adjuvant chemo. Completion of radiation therapy prior to registration is allowed, but it is preferred that patients receive radiation after randomization; patients randomized to pembrolizumab will receive their XRT concomitant with pembrolizumab. Adequate organ functions: ANC > 1.5, PLT > 100, Hgb > 9, normal creatinine, Tbili < 1.5 IUNL, AST/ALT/AlkPhos < 2.5 IULN. HIV with good CD4 count is allowed. Active autoimmune disease, Hep B,C, prior immunotherapy, active immunosuppressive therapy, or live vaccines within 30 days of registration are not allowed. Five unstained slides for PDL1 staining are required for stratification. The study has a dual primary endpoint; comparison of IDFS between arms in (i) all randomized patients (1-sided a=0.01) and in PDL-1 positive patients (1-sided a=0.015). Secondary endpoints include toxicity, overall survival, distant recurrence free survival (DRFS) and quality of life measures. Patients will be randomized 1:1 with stratification for PDL1 status, T size, nodal status and adjuvant chemo (yes or no) to observation or 1 year of pembrolizumab 200mg IV q 3 weeks. The accrual goal is N=1000 patients with estimated trial duration of 8 years. Two interim analyses are planned for all randomized patients when 50% and 75% of IDFS events have occurred for early stopping for either futility or efficacy. The study was activated on 11/15/16 and 34 patients were registered as of June 9, 2017. Cancer Trials Support Unit (CTSU) sites can use “OPEN” (https://open.ctsu.org) to enroll patients to this trial. Funding: NIH/NCI U10CA180888, U10CA180819, CA180868; and in part by Merck, Sharpe & Dohme, Corporation. Citation Format: Pusztai L, Barlow WE, Ganz PA, Henry NL, White J, Jagsi R, Mammen JMV, Lew D, Mejia J, Karantza V, Aktan G, Sharon E, Korde L, Hortobagyi GN, Mamounas E. SWOG S1418/NRG -BR006: A randomized, phase III trial to evaluate the efficacy and safety of MK-3475 as adjuvant therapy for triple receptor-negative breast cancer with > 1 cm residual invasive cancer or positive lymph nodes (>pN1mic) after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr OT1-02-04.

Long-term survival outcomes of triple-receptor negative breast cancer survivors who are disease free at 5 years and relationship with low hormone receptor positivity

Background:We counsel our triple-negative breast cancer (TNBC) patients that the risk of recurrence is highest in the first 5 years after diagnosis. However, there are limited data with extended follow-up on the frequency, characteristics, and predictors of late events.Methods:We queried the MD Anderson Breast Cancer Management System database to identify patients with stage I–III TNBC who were disease free at 5 years from diagnosis. The Kaplan–Meier method was used to estimate yearly recurrence-free interval (RFI), recurrence-free survival (RFS), and distant relapse-free survival (DRFS), as defined by the STEEP criteria. Cox proportional hazards model was used to compute hazard ratios (HRs) and 95% confidence intervals (CIs).Results:We identified 873 patients who were disease free at least 5 years from diagnosis with median follow-up of 8.3 years. The 10-year RFI was 97%, RFS 91%, and DRFS 92%; the 15-year RFI was 95%, RFS 83%, and DRFS 84%. On a subset of patients with oestrogen receptor and progesterone receptor percentage recorded, low hormone receptor positivity conferred higher risk of late events on multivariable analysis for RFS only (RFI: HR=1.98, 95% CI=0.70–5.62, P-value=0.200; RFS: HR=1.94, 95% CI=1.05–3.56, P-value=0.034; DRFS: HR=1.72, 95% CI=0.92–3.24, P-value=0.091).Conclusions:The TNBC survivors who have been disease free for 5 years have a low probability of experiencing recurrence over the subsequent 10 years. Patients with low hormone receptor-positive cancers may have a higher risk of late events as measured by RFS but not by RFI or DRFS.

Salivary proline rich peptide decreases cell growth in HCC38 triple negative breast cancer cell line

Objective: The objective of this study is to determine the effects of p1978 on the growth rate of a triple receptor negative breast cancer cell line.Methods: Three cell lines, 185B5 normal tissue, HCC38, and AU585, were seeded with peptide p1978. Corresponding plates were seeded with PBS to serve as controls. Baseline line cell counts were taken at 30% confluence prior to seeding. Counts were again made 48 hours later.Results: The HCC38 cell line showed decreased growth when exposed to the low and high dose of p1978 peptide.Conclusion: The results suggest that p1978 may have potential in treating triple receptor negative breast cancer.

A Synthetic Cell-Penetrating Dominant-Negative ATF5 Peptide Exerts Anticancer Activity against a Broad Spectrum of Treatment-Resistant Cancers.

Despite significant progress in cancer research, many tumor entities still have an unfavorable prognosis. Activating transcription factor 5 (ATF5) is upregulated in various malignancies and promotes apoptotic resistance. We evaluated the efficacy and mechanisms of the first described synthetic cell-penetrating inhibitor of ATF5 function, CP-d/n-ATF5-S1. Preclinical drug testing was performed in various treatment-resistant cancer cells and in vivo xenograft models. CP-d/n-ATF5-S1 reduced the transcript levels of several known direct ATF5 targets. It depleted endogenous ATF5 and induced apoptosis across a broad panel of treatment-refractory cancer cell lines, sparing non-neoplastic cells. CP-d/n-ATF5-S1 promoted tumor cell apoptotic susceptibility in part by reducing expression of the deubiquitinase Usp9X and led to diminished levels of antiapoptotic Bcl-2 family members Mcl-1 and Bcl-2. In line with this, CP-d/n-ATF5-S1 synergistically enhanced tumor cell apoptosis induced by the BH3-mimetic ABT263 and the death ligand TRAIL. In vivo, CP-d/n-ATF5-S1 attenuated tumor growth as a single compound in glioblastoma, melanoma, prostate cancer, and triple receptor-negative breast cancer xenograft models. Finally, the combination treatment of CP-d/n-ATF5-S1 and ABT263 significantly reduced tumor growth in vivo more efficiently than each reagent on its own. Our data support the idea that CP-d/n-ATF5-S1, administered as a single reagent or in combination with other drugs, holds promise as an innovative, safe, and efficient antineoplastic agent against treatment-resistant cancers. Clin Cancer Res; 22(18); 4698-711. ©2016 AACR.

Imaging and clinicopathological characteristics of breast cancer among women under the age of 40 years

Background: although breast cancer in young women is less common and often overlooked, it is still considered a major health concern.Objectives: to evaluate the demographic, clinical, radiological and histopathological characteristics of breast cancer among a sample of Iraqi women diagnosed under the age of 40 years.Patients and methods: a retrospective study enrolled 73 females below the age of 40 years with a history of breast cancer. All data was extracted from an established information system database designed by the Principal Investigator of the Iraqi National Breast Cancer Research Project under supervision of the International Agency for Research on Cancer (IARC) over a 4-years period from 2011 to 2014.Results: seventy three women under the age of 40 years with breast cancer were included in the study out of 625 diagnosed cases at different ages (11.68%). The age at presentation ranged from 25 to 39 years, the peak age frequency (63%) was recorded in the period between 35-39 years (n=46). Family history of breast cancer was noticed in approximately 31.5% of cases (n=23). Early menarche (less than 12 years old) was detected in 78.1% (n=57) of cases. On examination, 90.4% (n=66) of patients had painless palpable breast lump. By Sonography, the lesions were spiculated margins in 52% (n=38) of patients and echogenic halo around the mass was detected in 57.5% (n=42) of cases. Stage-II disease was recognized in 54.8%(n=40) of sample of the study and majority of the tumor (98.6%) were ductal in origin. According to Scarf- Bloom-Richardson method, 60.3% (n=44) of the tumors were moderately differentiated (i.e. grade- II). Eight patients (10.9%) were presented with triple receptor-negative breast cancer and recurrence of tumor was encountered in 17.8% (n=13) of cases.Conclusions: The current study suggest that breast cancer among young Iraqi women under 40 years had high incidence rate but might be less aggressive than what is reported in western countries despite of high recurrence rate.

Imaging and clinicopathological characteristics of breast cancer among women under the age of 40 years

Background: although breast cancer in young women is less common and often overlooked, it is still considered a major health concern.Objectives: to evaluate the demographic, clinical, radiological and histopathological characteristics of breast cancer among a sample of Iraqi women diagnosed under the age of 40 years.Patients and methods: a retrospective study enrolled 73 females below the age of 40 years with a history of breast cancer. All data was extracted from an established information system database designed by the Principal Investigator of the Iraqi National Breast Cancer Research Project under supervision of the International Agency for Research on Cancer (IARC) over a 4-years period from 2011 to 2014.Results: seventy three women under the age of 40 years with breast cancer were included in the study out of 625 diagnosed cases at different ages (11.68%). The age at presentation ranged from 25 to 39 years, the peak age frequency (63%) was recorded in the period between 35-39 years (n=46). Family history of breast cancer was noticed in approximately 31.5% of cases (n=23). Early menarche (less than 12 years old) was detected in 78.1% (n=57) of cases. On examination, 90.4% (n=66) of patients had painless palpable breast lump. By Sonography, the lesions were spiculated margins in 52% (n=38) of patients and echogenic halo around the mass was detected in 57.5% (n=42) of cases. Stage-II disease was recognized in 54.8% (n=40) of sample of the study and majority of the tumor (98.6%) were ductal in origin. According to Scarf-Bloom-Richardson method, 60.3% (n=44) of the tumors were moderately differentiated (i.e. grade- II). Eight patients (10.9%) were presented with triple receptor-negative breast cancer and recurrence of tumor was encountered in 17.8% (n=13) of cases.Conclusions: The current study suggest that breast cancer among young Iraqi women under 40 years had high incidence rate but might be less aggressive than what is reported in western countries despite of high recurrence rate.

Death-associated protein kinase 1 promotes growth of p53-mutant cancers.

Estrogen receptor-negative (ER-negative) breast cancers are extremely aggressive and associated with poor prognosis. In particular, effective treatment strategies are limited for patients diagnosed with triple receptor-negative breast cancer (TNBC), which also carries the worst prognosis of all forms of breast cancer; therefore, extensive studies have focused on the identification of molecularly targeted therapies for this tumor subtype. Here, we sought to identify molecular targets that are capable of suppressing tumorigenesis in TNBCs. Specifically, we found that death-associated protein kinase 1 (DAPK1) is essential for growth of p53-mutant cancers, which account for over 80% of TNBCs. Depletion or inhibition of DAPK1 suppressed growth of p53-mutant but not p53-WT breast cancer cells. Moreover, DAPK1 inhibition limited growth of other p53-mutant cancers, including pancreatic and ovarian cancers. DAPK1 mediated the disruption of the TSC1/TSC2 complex, resulting in activation of the mTOR pathway. Our studies demonstrated that high DAPK1 expression causes increased cancer cell growth and enhanced signaling through the mTOR/S6K pathway; evaluation of multiple breast cancer patient data sets revealed that high DAPK1 expression associates with worse outcomes in individuals with p53-mutant cancers. Together, our data support targeting DAPK1 as a potential therapeutic strategy for p53-mutant cancers.