TNBC Cell Lines Research Articles

Abstract 1240: RRM2 and mutant p53 as therapeutic targets in TNBC

Abstract Triple negative breast cancer (TNBC) is a highly aggressive type of cancer associated with poor prognosis and characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor type 2 (HER2). TNBC accounts for 10 - 15% of all new cases of breast cancer. TNBC is heterogenous in nature (70% basal-like) and currently doesn't have effective targeted therapy. As such, dose-dense chemotherapeutic agents used in combination, such as doxorubicin (DOXO) with cyclophosphamide, is the regimen of choice for TNBC. However, DOXO has limited efficacy as a therapeutic agent owing to acquired drug resistance and its cardiotoxic effects. Ribonucleotide reductase (RR) is the rate limiting enzyme required for the conversion of ribonucleotide to deoxyribonucleotide in DNA synthesis during cell proliferation. Didox (3,4 dihydroxybenzohydroxamic acid) is a unique RR inhibitor with iron chelating and free radical scavenging properties. We have previously identified that ribonucleotide reductase M2 (RRM2) is upregulated in TNBC cells and is a key contributor to acquired drug resistance. We found that Didox not only inhibits RRM2, but it also reduces mutant p53 protein levels and inhibits the activation of the NF-κB pathway. Through this inhibition, we found that Didox works synergistically with DOXO to inhibit TNBC tumor growth while also minimizing DOXO-induced cardiac damage in mice. In our present study, we have observed that RRM2 expression is upregulated in TNBC patients from three independent data sets and that high RRM2 levels are associated with lower recurrence free survival (RFS) and overall survival (OS). Interestingly, high levels of RRM2 were only predictive of lower RFS and OS in patients with wild-type p53 but not mutant p53. Additionally, we have found that Didox downregulates mutant p53 protein levels in TNBC cell lines but does not alter total p53 in cells with functioning p53. Our data confirms that Didox induced downregulation of mutant p53 is not a transcriptional effect. Finally, we have identified that RRM2 protein levels increase in TNBC with progression of stage as measured in patient tissue microarray analysis. Our data presents a novel and promising approach for the treatment of TNBC that involves inhibition of RRM2, NF-kB activation, and mutant p53 that merits further clinical investigation in human models. Citation Format: Nahid Sultana, Elizabeth A. Wilson, Howard L. Elford, Jesika S. Faridi. RRM2 and mutant p53 as therapeutic targets in TNBC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1240.

Abstract 1881: AFM24 is a novel, highly potent, tetravalent bispecific EGFR/CD16A-targeting Innate Cell Engager (ICE®) designed for the treatment of EGFR-positive malignancies

Abstract AFM24 engages CD16A (FcγRIIIa) on NK cells and macrophages with much higher affinity than monoclonal antibodies (mAbs), triggering NK cell-mediated ADCC and macrophage-mediated ADCP. AFM24's mode of action is highly differentiated from current EGFR-targeting treatment options since it does not rely on inhibition of the EGFR signaling pathway, thus rendering AFM24 insensitive to the mutational status (e.g. KRAS, BRAF) of the tumor. Indeed, the efficacy of currently marketed EGFR inhibitors is limited by i) toxicities arising from blocking of EGFR signaling in healthy tissues and ii) by the occurrence of intrinsic or acquired resistances of the tumor to EGFR-targeting inhibitors. With its distinctive mode of action profile, AFM24 is a novel treatment option for a wide range of EGFR+ patient populations including hard-to-treat patients. In preclinical assays AFM24 added up to 20% more ADCC and up to 6-fold more ADCP towards NSCLC, CRC, HNSCC and ccRCC cell lines with a wide range of EGFR densities when compared to EGFR-targeting mAbs. This superior effect of AFM24 was most pronounced towards target cell lines with low EGFR density or EGFR signaling pathway mutations, such as KRAS. AFM24's efficacy was assessed in a xenograft mouse model using different human EGFR+ cell lines such as the TNBC cell line MDA-MB-231 and an adoptively transferred, cytokine-stimulated and expanded NK cell product. This combination of AFM24 with an NK cell product demonstrated a significant dose-dependent and differentiated reduction in tumor outgrowth as compared to mice treated with NK cell product alone. This model allows for systematic screening of future IO-combination regimens, such as checkpoint inhibitors. Moreover, prevalence and activity of innate and adaptive immune cells were assessed in tumor explants of patients with EGFR+ malignancies. These data will be instrumental to guide the clinical development of AFM24 in monotherapy and combination therapies. In conclusion and considering the previously reported good safety profile in cynomolgus monkeys, the presented data shows AFM24's potential to become a novel treatment option for patients with EGFR+ cancers overcoming limitations of available EGFR-targeting therapies by fully exploiting the potential of the innate immune system. AFM24 is currently being investigated in a phase 1/2a study, NCT04259450, in EGFR+ tumors. Citation Format: Jens Pahl, Gabriele Hintzen, Uwe Reusch, Torsten Haneke, Christian Breunig, Sheena Pinto, Cassandra Choe-Juliak, Andreas Harstrick, Wolfgang Fischer, Arndt Schottelius, Joachim Koch, Erich Rajkovic. AFM24 is a novel, highly potent, tetravalent bispecific EGFR/CD16A-targeting Innate Cell Engager (ICE®) designed for the treatment of EGFR-positive malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1881.

Abstract 943: Insulin-like growth factor-2 a potential target for screening and treatment in patients with triple negative breast cancer

Abstract Triple-negative breast cancer (TNBC) affects only about 10-15% of women with breast cancer (BC) but accounts for almost 50% of all BC deaths. At this time, development of targeted treatments for TNBC are urgently needed. Insulin-like growth factor-2 (IGF2) is highly expressed in TNBCs and mediates its proliferative effects via two hormone receptors, insulin-like growth factor -1 receptor (IGF1R) and insulin receptor (IR). Using a TNBC tissue microarray (TMA) consisting of 159 archival clinical samples, we confirm that IGF2 is highly expressed in tumor tissue as compared to nearby normal tissue (P<0.001). In addition, evaluation of a second breast cancer TMA that included archival TNBC samples from African American and Latino women, IGF2 expression was similarly higher in these samples than in samples from Caucasian women (P<0.02). Since enrichment of IGF1R and IR are detected in TNBC and are known to allow signaling crosstalk, the effects of BMS-754807, a dual IGF1R/IR antagonist, were assessed on TNBC progression in vitro and in vivo with human and murine models. To determine the effects of BMS-754807 in vitro, we used cell viability assays with TNBC cell lines, MDA-MB-231 and HCC1806. BMS-754807 significantly reduced cell viability after 48 and 72 hours (P< 0.01), and a similar effect was observed in the murine TNBC cell line 4T1 (P< 0.01). In vivo, BMS-754807 inhibited tumor growth of human TNBC as well as mouse 4T1 xenografts (P< 0.05). Further, in studies of immune cell populations using syngeneic 4T1 tumor xenografts, BMS-754807 significantly decreased myeloid derived suppressor cells (P <0.05) in the tumor microenvironment (TME) and in the spleen of BALC/c mice. Additionally, there was an increase in T effector CD8+ and CD4+ tumor infiltrating lymphocytes (P< 0.05), particularly of spleen T central memory cells (P<0.01). Notably, conversion of CD8+ from T central memory to a T effector memory phenotype that is fully active against tumors can confer long term immunity against recurrence. This data suggest that IGF2 is a potential biological marker for breast cancer disparities and that targeting receptors of IGF2 with drugs such as BMS-754807 could provide additional treatment options in combination with current therapies, thus reducing TNBC progression and ultimately improving patient outcomes. [Funded by NIH U54 143931 Cancer Center Partnership and CBCRP] Citation Format: Nalo M. Hamilton, Diana Marquez-Garban, David Austin, Neda Moatamed, Lorena P. Burton, David Elashoff, Begonya Comin-Anduix, Tristan Grogan, Madhuri Wadehra, Helena Chang, Adrienne Martinez, Mary-Lynn Brecht, Isabel Jabara, Richard Pietras, Yahya Elshimali, Jaydutt Vadgama, Michael Jung. Insulin-like growth factor-2 a potential target for screening and treatment in patients with triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 943.

Abstract 1874: MDX-124, a novel annexin-A1 antibody, induces an anti-tumor immune response and wide-ranging anti-cancer activity in multiple preclinical models

Abstract Major recent advances have increased the understanding of both the molecular mechanisms and the immune response in cancer. This has led to the discovery of new agents that regulate the tumor immune microenvironment. Annexin-A1 (ANXA1) is a phospholipid binding protein that has been shown to have immunomodulatory effects on T-cells, macrophages and dendritic cells. Although normally localized in the cytoplasm, ANXA1 is secreted in response to a number of different stimuli and can modulate cellular functions through interactions with formyl peptide receptors (FPR1/2). Overexpression of ANXA1 has been observed in several cancer types including triple-negative breast (TNBC), lung, pancreatic and ovarian, and correlates with poor prognosis and decreased overall survival. Furthermore, ANXA1 has also been shown to influence cancer cell proliferation, angiogenesis, migration and drug resistance. Hence, we explored the role of MDX-124, a novel humanized antibody targeting ANXA1, and here present data from several pre-clinical cancer models. Results: Incubation of a panel of cell lines representing several histological cancer subtypes with MDX-124 for 72h resulted in a statistically significant dose-dependent reduction in cell viability compared to control. ANXA1 expression in the subcellular compartments (nucleus, cytoplasm and membrane) was quantified in a panel of cancer cell lines via imaging flow cytometry and the anti-proliferative effect of MDX-124 was shown to correlate with membrane ANXA1 expression. Using a transwell assay, TNBC and acute myeloid leukemia cell lines exposed to MDX-124 exhibited a statistically significant reduction in migration compared to untreated controls. We also observed that MDX-124 induced antibody dependent cellular cytotoxicity (ADCC) activity in a dose-dependent manner using a reporter bioassay (EC50 = 0.38 nM). MDX-124 was further evaluated in an additional panel of in-vitro assays and shown to exert consistent anti-cancer activity. In conclusion, our data indicate that targeting ANXA1 with MDX-124 inhibits tumorigenic processes and induces an immune response in tumors overexpressing ANXA1. MDX-124 therefore provides an innovative approach to cancer therapy. Investigations are continuing to explore the efficacy of MDX-124 in syngeneic mouse models as both a single agent and in combination with standard of care agents. Citation Format: Fiona C. Dempsey, Hussein Al-Ali, Scott J. Crichton, Chris Pepper, Christopher N. Parris. MDX-124, a novel annexin-A1 antibody, induces an anti-tumor immune response and wide-ranging anti-cancer activity in multiple preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1874.

Abstract 1403: Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss

Abstract Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC is an aggressive subtype of breast cancer that lacks the expression of estrogen and progesterone receptors and HER2 protein. This lack of functioning receptors results in limited treatment options, usually resulting in the use of taxanes, platinums, and anthracyclines. Although TNBC often starts as being sensitive to traditional chemotherapies, tumor relapse and/or resistance often develops. Paclitaxel (PTX) is a commonly used taxane for TNBC, well known to induce mitotic arrest through stabilization of microtubules (MT) leading to an interference with the treadmilling function of MT through the activation of the spindle assembly checkpoint. We have previously shown that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor significantly increases resistance to PTX in APC knockdown (APCKD) cells generated in two human TNBC cell lines, MDA-MB-157 and MDA-MB-231. Given this striking PTX resistance, we were interested in seeing if resistance in APCKD can be seen using other non-taxane MT targeting agents. To accomplish this, we have used an epothilone (Epothilone B) and a vinca alkaloid (Vinblastine), that function by stabilizing and destabilizing the MTs, respectively. Our previous investigations demonstrated that APC loss results in increased CDK1 and CDK6, so these have been used as markers of cell cycle progression after drug treatment. In addition, changes in apoptosis were measured through the use of Annexin V and PI staining, and cleaved caspase 3 expression. Examined together, a better understanding of how APC loss influences chemoresistance in TNBC can lead to more efficient treatments for TNBC patients resistant to taxanes. Given the critical interaction between APC and MTs, this may be an avenue for targeted therapies to overcome taxane resistance. Citation Format: Pamela D. Arenas, Sara M. Maloney, Jenifer R. Prosperi. Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1403.

Abstract 1977: Combined inhibition of EGFR and PI3Kinase signaling in EGFR amplified triple negative breast cancer cells induces apoptosis and reduces cancer stem cells

Abstract Signaling from the Epidermal Growth Factor Receptor (EGFR) family is commonly dysregulated in various cancer types by amplification or activating mutations. For example, HER2 amplification occurs in 15-20% of breast cancers, and is a well-defined driver of tumorigenesis. EGFR is amplified in approximately 2% of breast cancer patients with unknown significance. It is amplified more frequently in TNBC (5.7-6.7%) and ER-/HER2+ cancers (5.8-6.5%). Basal (4.3-8.1%) and HER2 enriched (3.9-45.4%) molecular subtypes show the highest amplification than other molecular subtypes. EGFR amplification leads to shorter overall survival among breast cancer patients compared to those without amplified EGFR. Approximately 71% of EGFR amplified tumors have activated PI3K signaling, indicating a possible dual-inhibition therapeutic approach targeting both EGFR and PI3K. In order to study the role of EGFR amplification and PI3K aberrations in TNBC, we used the TNBC cell lines BT20 (amplified EGFR, PIK3CA activating mutation), MDA-MB-468 (amplified EGFR, PTEN deletion), and MDA-MB-231 (no amplification of EGFR or PI3K pathway mutations). EGFR amplification in both MDA-MB-468 and BT20 cell lines was confirmed by western blotting and Fluorescent in situ Hybridization; MDA-MB-231 did not have EGFR amplification. Activating mutations in both alleles of PIK3CA in BT20 was confirmed by sequencing. Loss of PTEN in MDA-MB-468 was confirmed by immunoblotting. Inhibition of EGFR with erlotinib resulted in decreased ERK signaling in BT20 and MDA-MB-468, but not in MDA-MB-231. Inhibition of the PI3K with Ly294002 or BKM120 decreased activation of AKT and mTOR signaling in BT20 and MDA-MB-468, but not in MDA-MB-231. The combination of erlotinib and PI3K inhibitors more dramatically reduced mTOR and AKT signaling in the BT20 and MDA-MB-468 cells. Combination of erlotinib and PI3K inhibitors reduced cell viability in these 3 cell lines, but inhibition was greater in MDA-MB-468 and BT20 compared to MDA-MB-231, and only MDA-MB-468 and BT20 cells had an increased fraction of apoptotic cells, signifying that combination therapy is most effective in cells with amplified EGFR and aberrant PI3K. It has been previously shown that HER2/HER3 activation plays a role in self renewal of tumor initiating cells. We demonstrate that the combination of erlotinib and BKM120 reduced the cancer stem cell population as measured by an aldeflour assay and by mammosphere formation. In summary, these data demonstrate that EGFR amplification in TNBC drives cell viability through EGFR and PI3K signaling, and EGFR/PI3K inhibitor combination causes apoptosis as well as reduction in the cancer stem cell population. We present evidence therefore that dual inhibition of EGFR/PI3K presents as a potential targetable pathway in TNBC with EGFR amplification and aberrant PI3K signaling. Citation Format: David John Wisniewski, Donna Voeller, Stanley Lipkowitz. Combined inhibition of EGFR and PI3Kinase signaling in EGFR amplified triple negative breast cancer cells induces apoptosis and reduces cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1977.

Abstract 3116: Designing a model system for the study of CD44-Ezrin interactions in breast cancer progression and drug resistance

Abstract Triple-negative breast cancer (TNBC) has the highest risk of relapse and is thought to be the most aggressive subtype of BC. This subtype is known to express high levels of the cancer stem cell (CSC) marker CD44. The tumor-initiating properties of CSC's, as well as their resistance to chemotherapy, contribute to the difficulty in successfully treating TNBCs.CD44 is a transmembrane glycoprotein that mediates communication between cells and the extracellular matrix. In doing so, it acts as a central node in several cancer-related signaling pathways through interactions with binding partners such as Ezrin to promote tumor growth, invasion and survival. Furthermore, expression of CD44 is associated clinically with positive lymph node status, recurrence and poor overall survival in BC. We aimed to validate CD44's role in these cancer promoting properties and develop a genetically tractable and engraftable TNBC cell model to study CD44 in the context of interactions with the Ezrin adaptor protein. We first hypothesize that disrupting CD44 expression will attenuate the metastatic and drug resistant potential of TNBC cells. To address this, we utilized CRISPR-Cas9 gene editing to knockout (KO) CD44 expression in the MDA-MB-231 human TNBC cell line and then subjected CD44 KO cells, KO cells rescued with wild type CD44, or the parental MDA-MB-231 cells to a variety of in vitro assays to assess proliferative, migratory and drug resistant potential, as well as mouse lung metastatic seeding in vivo. We show that the transmembrane glycoprotein CD44 plays an important role in cancer cell proliferation, migration, and chemotherapeutic resistance in vitro as well as having an essential role in metastatic seeding in vivo in the MDA-MB-231 cell model of TNBC. Our results validate the utility of this CRISPR-Cas9 KO and rescue TNBC cell model for the study of CD44 involvement in drug resistance and metastasis. More importantly we have set the stage for further investigating CD44's role in the context of interactions with the Ezrin adaptor protein. Citation Format: Rayanna Jane Birtch, Peter Greer, Yan Gao, Victoria Hoskin. Designing a model system for the study of CD44-Ezrin interactions in breast cancer progression and drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3116.

Inhibitors of PD-1/PD-L1 and ERK1/2 impede the proliferation of receptor positive and triple-negative breast cancer cell lines

PurposeTriple-negative breast cancer (TNBC) is characterized by an unfavorable prognosis and missing systemic therapeutic approaches beside chemotherapy. Targeting the immune checkpoint PD-1/PD-L1 showed promising results in breast cancer and especially in TNBC. The extracellular signal-regulated kinase 1/2 (ERK1/2) is an important driver of carcinogenesis. Here, the effect of combined PD-1/PD-L1 and ERK1/2 inhibitor treatment is investigated of cell growth and intracellular impact of breast cancer cell lines.MethodsThe IC50 values of each inhibitor and the effect of combined treatment were determined in three TNBC cell lines of different subtypes and one non-TNBC cell line. Phospho-specific antibodies were used in western blot analyses to investigate an effect on ERK1/2 activation. Expressions of immune modulatory and cell cycle-associated genes were examined by quantitative reverse transcription PCR.ResultsBoth inhibitors PD-1/PD-L1 and ERK1/2 impeded the proliferation of TNBC to a higher extent than of non-TNBC. By combined treatment, cell lines were inhibited either synergistically or additively. ERK1/2 and S6 phosphorylation were reduced and expressions of c-Fos and FosL were diminished after ERK1/2 inhibitor as single and combined treatment. Between genes involved in immune modulation, IL-8 was upregulated in TNBC cells after combined treatment.ConclusionIn conclusion, combination of PD-1/PD-L1 and ERK1/2 inhibitors showed favorable effects for a new therapy strategy, with better results in TNBC cell lines than in non-TNBC cells. The effects have to be validated in models that can reflect the interaction between immune and tumor cells like the situation in the tumor micro-environment.

LncRNA CARMN overexpression promotes prognosis and chemosensitivity of triple negative breast cancer via acting as miR143-3p host gene and inhibiting DNA replication

BackgroundTriple negative breast cancer (TNBC) is a subtype of breast cancer with poor prognosis and lack of effective treatment target. Here we screened differentially expressed lncRNAs through bioinformatics analysis and identified CARMN as a downregulated lncRNA which is lowest expressed in TNBC. We aimed to identify the potential role and molecular mechanisms of CARMN in TNBC.MethodsPredictive value of CARMN was explored in breast cancer cohorts. TNBC cell lines with CARMN overexpression or CARMN silence and were used for in vitro and in vivo experiments. RNA-seq of CARMN overexpressed cells was performed for exploring downstream of CARMN.ResultsCARMN is downregulated at different phase of malignant transformation of breast tissue. CARMN can predict both better prognosis and higher response rate of cisplatin-based neoadjuvant chemotherapy in breast cancer. A nomogram is built to predict cisplatin-based chemotherapy response in breast cancer. Through in vitro and in vivo studies, we confirmed CARMN can also inhibit tumorigenesis and enhance sensitivity to cisplatin in TNBC cells. RNA-seq and further experiments revealed CARMN can inhibit DNA replication. MCM5, an important DNA replication initiation factor, is the most downregulated gene in DNA replication pathway following CARMN overexpression. We confirmed CARMN can produce miR143-3p from its exon5 which is DROSHA and DICER dependent, resulting binding and decrease of MCM5. Moreover, suppressing miR143-3p can weaken function of CARMN in suppressing tumorigenesis and promoting chemosensitivity.ConclusionsOur results indicated lncRNA CARMN is a predictive biomarker of better prognosis and enhanced cisplatin sensitivity in TNBC. CARMN is the host gene of miR143-3p which downregulates MCM5, causing inhibited DNA replication.

Depleting adenine to prime for apoptosis

Cancer Triple-negative breast cancer (TNBC) is difficult to treat. Daniels et al. identified a metabolic vulnerability in TNBC patient cells and cell lines that may create a therapeutic opportunity for patients. The authors searched for metabolism-perturbing small-molecule compounds that sensitized TNBC lines to apoptosis induced by BH3 mimetics that block anti-apoptotic proteins. They found that inhibition of the rate-limiting enzyme of the nicotinamide adenine dinucleotide salvage pathway, specifically the loss of adenine downstream of inhibition, primed TNBC cells for apoptotic death. Sci. Signal. 14 , eabc7405 (2021).

Inhibition of triple negative breast cancer metastasis and invasiveness by novel drugs that target epithelial to mesenchymal transition

Invasive breast cancer (BrCa) is predicted to affect 1 in 9 women in a lifetime;1 in 32 will die from this disease. The most aggressive forms of BrCa, basal-like/triple-negative phenotype (TNBC), are challenging to treat and result in higher mortality due high number of metastatic cases. There is a paucity of options for TNBC treatment, which highlights the need for additional innovative treatment approaches. NIH-III mice were injected in the abdominal mammary fat pad with luciferase-expressing derivative of the human TNBC cell line, MDA-MB-231 cells. Animals were gavage-fed with nitrofen at the doses of 1, 3 or 6 mg/kg/alternate days. However, several structural properties/components of nitrofen raise concerns, including its high lipophilicity (cLogP of nearly 5) and a potential toxophore in the form of a nitroarene group. Therefore, we developed analogues of nitrofen which lack the nitro group and/or have replaced the diaryl ether linker with a diarylamine that could allow modulation of polarity. In vitro anti-invasiveness activity of nitrofen analogues were evaluated by quantitative determination of invasion of MDA-MB-231-Luciferase cells through Matrigel using a Boyden chamber. Our in vivo data show that nitrofen efficiently blocks TNBC tumor metastasis. In vitro data suggest that this is not due to cytotoxicity, but rather is due to impairment of invasive capacity of the cells. Further, using an in vitro model of EMT, we show that nitrofen interferes with the process of EMT and promotes mesenchymal to epithelial transformation. In addition, we show that three of the nitrofen analogues significantly reduced invasive potential of TNBC cells, which may, at least partially, be attributed to the analogues’ ability to promote mesenchymal to epithelial-like transformation of TNBC cells. Our study shows that nitrofen, and more importantly its analogues, are significantly effective in limiting the invasive potential of TNBC cell lines with minimal cytotoxic effect. Further, we demonstrate that nitrofen its analogues, are very effective in reversing mesenchymal phenotype to a more epithelial-like phenotype. This may be significant for the treatment of patients with mesenchymal-TNBC tumor subtype who are well known to exhibit high resistance to chemotherapy.

KIF23 promotes triple negative breast cancer through activating epithelial-mesenchymal transition.

KIF23 is a member of kinesin family, recent researches indicate KIF23 plays an important role in the proliferation and migration of malignant cancer cells. While the function and specific molecule mechanism of KIF23 in triple negative breast cancer remains unclear. QRT-PCR and immunohistochemistry were conducted to analyze expression of KIF23 in triple negative breast cancer tissues and paired paracancer tissues. CCK-8 assay, colony formation assay, wound healing assay and transwell assay were applied for exploring phenotype changing of triple negative breast cancer cell lines MDA-MB-231 and BT549 after siRNA-induced knockdown of KIF23. Several bioinformatic databases were used for predicting miRNAs that combing with KIF23 mRNA and verified by dual luciferase reporter assay. Western blot assay was performed to explore downstream signaling pathway of KIF23. KIF23 was overexpressed in triple negative breast cancer, knockdown of KIF23 by siRNA inhibited proliferation and migration of TNBC cell lines MDA-MB-231 and BT549. Mechanistically, knockdown of KIF23 resulted in the suppression of Epithelial-Mesenchymal Transition. Meanwhile, miR-195-5p was downregulated in TNBC, and dual luciferase reporter assay indicated miR-195-5p could combine with 3'UTR of KIF23 thus promoting degradation of KIF23. KIF23 is a potential oncogene in triple negative breast cancer, miR-195-5p could combine with 3'UTR of KIF23. Our study reveals a new sight into triple negative breast cancer.

Combinatorial targeting of microRNA-26b and microRNA-101 exerts a synergistic inhibition on cyclooxygenase-2 in brain metastatic triple-negative breast cancer cells.

Extravasation of triple-negative (TN) metastatic breast cancer (BC) cells through the brain endothelium (BE) is a critical step in brain metastasis (BM). During extravasation, metastatic cells induce alteration in the inter-endothelial junctions and transmigrate through the endothelial barrier. Transmigration of metastatic cells is mediated by the upregulation of cyclooxygenase-2 (COX-2) that induces matrix metalloproteinase-1 (MMP-1) capable of degrading inter-endothelial junctional proteins. Despite their important role in BM, the molecular mechanisms upregulating COX-2 and MMP-1 in TNBC cells remain poorly understood. In this study, we unraveled a synergistic effect of a pair of micro-RNAs (miR-26b-5p and miR-101-3p) on COX-2 expression and the brain transmigration ability of BC cells. Using a gain-and-loss of function approach, we modulated levels of miR-26b-5p and miR-101-3p in two TNBC cell lines (the parental MDA-MB-231 and its brain metastatic variant MDA-MB-231-BrM2), and examined the resultant effect on COX-2/MMP-1 expression and the transmigration of cancer cells through the BE. We observed that the dual inhibition of miR-26b-5p and miR-101-3p in BC cells results in higher increase of COX-2/MMP-1 expression and a higher trans-endothelial migration compared to either micro-RNA alone. The dual restoration of both micro-RNAs exerted a synergistic inhibition on COX-2/MMP-1 by targeting COX-2 and potentiated the suppression of trans-endothelial migration compared to single micro-RNA. These findings provide new insights on a synergism between miR-26-5p and miR-101-3p in regulating COX-2 in metastatic TNBC cells and shed light on miR-26-5p and miR-101-3p as prognostic and therapeutic targets that can be exploited to predict or prevent BM.

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

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

LncRNA SNHG6/miR-125b-5p/BMPR1B Axis: A New Therapeutic Target for Triple-Negative Breast Cancer.

BackgroundTriple-negative breast cancer (TNBC) is a significant cause of patient morbidity. The exactly pathobiological features of this condition has yet to be completely elucidated.MethodsBreast cancer data obtained from The Cancer Genome Atlas (TCGA) database were evaluated for lncRNA SNHG6 expression. Normal human breast epithelial cell line (MCF-10A) and other breast cancer cell lines (BT-549, MDA-MB-231, Hs 578t, ZR-75-30, SK-BR-3, MCF-7) were also assessed for lncRNA SNHG6 expressions. Cellular proliferative ability was evaluated with colony formation and CCK-8 assays. The ability of cells to migrate was scrutinized with the wound healing and Boyden chamber cell migration assays. qRT-PCR enabled for detection of lncRNA SNHG6, miR-125b-5p and BMPR1B mRNA expressions. Protein BMPR1B expressions were further assessed using Western Blotting. Direct binding sites between transcripts were determined using dual-luciferase reporter assays. We also constructed a xenograft mouse model to further dissect the vivo implications of lncRNA SNHG6. Ki-67 and c-Caspase-3 expressions were detected using immunohistochemistry staining.ResultsBreast cancer cell lines demonstrated higher lncRNA SNHG6 expressions, particularly TNBC cell lines, in contrast to normal breast epithelial cell lines. This finding coincided with those noted on analysis of TCGA breast cancer data. lncRNA SNHG6 knockdown inhibited TNBC cell proliferation, migration, while promoted cell apoptosis. Furthermore, suppressed lncRNA SNHG6 expressions resulted in lower tumor weights and volumes in a xenograft mouse model, as evidenced by Ki-67 and c-Caspase-3 expression profiles in tumor tissues. miR-125b-5p and lncRNA SNHG6/BMPR1B both possessed direct binding sites for each other which was validated utilizing a dual-luciferase reporter assay. Decreasing lncRNA SNHG6 expression in TNBC cells upregulated miR-125b-5p expression. Another side, inhibiting miR-125b-5p upregulated BMPR1B expression in these cells. Moreover, knocking down lncRNA SNHG6 downregulated BMPR1B expression in TNBC cells, and the finding was rescued in cells which were exposed to miR-125b-5p inhibitor. Downregulating miR-125b-5p mitigated the effect of suppressing lncRNA SNHG6 on TNBC cell proliferation, migration, and apoptosis.ConclusionDownregulation of lncRNA SNHG6 could inhibit TNBC cell proliferative, migratory capabilities and promote apoptosis capability, likely through modulation of the miR-125b-5p/BMPR1B axis. This axis may be targeted in formulating new therapies for TNBC.

Androgen Receptor Highjacks ErbB-2 Nuclear Function to Induce Triple Negative Breast Cancer Growth

Triple negative breast cancer (TNBC) has poor prognosis and neither established biomarkers nor therapeutic targets. On the one hand the androgen receptor (AR), a steroid hormone receptor (SR) which is expressed in 10-53% of TNBC and proved to be critical for BC proliferation, has been proposed as a new target in TNBC. On the other hand, we and others have shown that membrane ErbB-2 migrates to the nucleus (nuclear ErbB-2, NErbB-2) where it binds DNA at HER-2 associated sequences (HAS) to regulate BC proliferation and migration. Since we have previously shown a functional interplay between growth factors and SR signaling pathways in BC, we propose the existence of an interaction between AR and ErbB-2 which is involved in NErbB-2+/AR+ BC growth. The experimental model used was the human TNBC cell line MDA-MB-453 which displays high expression levels of AR and NErbB-2. By Western Blot (WB) we found that dihydrotestosterone (DHT) treatment for short times (minutes) did not regulate ErbB-2 phosphorylation status at residues Tyr1221/1222 and 1248 which were constitutively activated. However, DHT led to an increase in ErbB-2 phosphorylation at residue Tyr877 which we have proved to be required for ErbB-2 nuclear migration. The latter effect was blocked by the AR antagonist enzalutamide (enza). Blockage of Src activity with dasatinib inhibited DHT-induced ErbB-2 phosphorylation at Tyr877. By Immunofluorescence and confocal microscopy analyses and subcellular fractionation studies we demonstrated that DHT induced ErbB-2 nuclear migration which was inhibited by enza. By chIP we found that DHT induced ErbB-2 recruitment to a HAS site in ERK5, a gene involved in BC proliferation, and to a HAS site in FKBP5, a classical AR responsive gene. By WB we demonstrated that transfection with an ErbB-2 mutant which is unable to translocate to the nucleus and functions as a dominant negative inhibitor of ErbB-2 nuclear migration (hErbB-2ΔNLS), inhibited FKBP51 up-regulation by DHT. Finally, by microarray and bioinformatics analysis we identified 315 differentially expressed genes (DEGs) in the presence of DHT and NErbB-2 eviction. Enrichment analyses showed that the DEGs belonged to the immune response and interferon pathways. Kaplan-Meier analysis revealed that the expression of 6 genes was significantly associated with overall survival in TNBC patients from the METABRIC cohort: CXCL10, TAP1, STAT1, NMI, HLA-A and NLRC5. Multivariate Cox regression analysis identified the combined expression of the 6 genes as an independent predictor of better clinical outcome in TNBC (HR: 0.56, 95% CI 0.38-0.82, P = 0.003). In conclusion, our findings evidence that DHT-activated AR induces Src-mediated ErbB-2 rapid activation and its migration to the nucleus where it binds to HAS sites in the DNA. Moreover, based on the DEGs of NErbB-2 eviction in presence of DHT we identified a gene signature associated with favorable outcome in TNBC.

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

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

Molecular Docking Study of α-Cyclodextrin With Psoralen: MDA-MB-231 Cancer Cell

Psoralen is an important bioactive component, isolated from the leaves of Ficus carica (Fig). Psoralen is proved to inhibit breast cancer cell growth and in tumor bearing mice the function of osteoblasts and osteoclasts is regulated. It is difficult to treat Triple-Negative Breast Cancer, a sub type of breast cancer. On treating Fig leaf extract, it is found that the leaf extract inhibits the proliferation of MDA-MB-231, which is a TNBC cell line, but not MCF10A cells, which is normal breast epithelial cell line. To increase the stability, solubility, volatility, it is beneficial to encapsulate natural products with α-Cyclodextrin (α-CD). Cyclodextrin constituted by 6 glucose units are termed as α-Cyclodextrin. Compared to host molecule guest: host inclusion complexes exhibit improved chemical or biological properties. Main aim of the study is interaction of Psoralen with α-CD and MDA-MB-231 Cancer cell with α-CD: Psoralen inclusion complex by molecular docking studies. 3D structures of Psoralen, α-CD and MDA-MB-231 cancer cell are obtained and docking is carried out using PatchDock server. Model which is highly favorable will have a high score. Energetically favorable and most probable structures of α-CD: Psoralen and MDA-MB-231 cancer cell: α-CD Psoralen inclusion complex has a score of 1762 and 6230 respectively.

Anticancer effects of radiation therapy combined with Polo-Like Kinase 4 (PLK4) inhibitor CFI-400945 in triple negative breast cancer

Development of novel multimodality radiotherapy treatments in metastatic breast cancer, especially in the most aggressive triple negative (TNBC) subtype, is of significant clinical interest. Here we show that a novel inhibitor of Polo-Like Kinase 4 (PLK4), CFI-400945, in combination with radiation, exhibits a synergistic anti-cancer effect in TNBC cell lines and patient-derived organoids in vitro and leads to a significant increase in survival to tumor endpoint in xenograft models in vivo, compared to control or single-agent treatment. Further preclinical and proof-of-concept clinical studies are warranted to characterize molecular mechanisms of action of this combination and its potential applications in clinical practice.

Differential reprogramming of breast cancer subtypes in 3D cultures and implications for sensitivity to targeted therapy

Screening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established.