Calcitonin Receptor is a Novel Biomarker for NF-κB-Mediated Cellular Stress in Triple-Negative Breast Cancer Cells.

Background: The abnormal expression of kinase family member 4A (KIF4A) is linked to breast cancer progression, with numerous miRNAs exhibiting abnormal expression. Thus, there is an urgent need to investigate the mechanisms of action of miRNAs and their target genes for the diagnosis and treatment of breast cancer. Materials and Methods: A bioinformatics analysis was conducted to screen for KIF4A, a key gene involved in oxidative stress in breast cancer cells. Using CCK8, EdU, cell healing, and Transwell assays, the knockdown of KIF4A was found to effectively inhibit the proliferation, migration, and invasion of breast cancer cells. Dual-luciferase assay and Western blotting confirmed that miR-223-3p targets and regulates KIF4A expression. The impact of miR-223-3p and KIF4A on oxidative stress in breast cancer cells was assessed through reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) measurements. Flow cytometry was used to evaluate tumor cell apoptosis. Results: Our results suggest that KIF4A is a downstream target of miR-223-3p. miR-223-3p inhibits the proliferation and invasion of breast cancer cells by directly targeting and downregulating KIF4A. Importantly, we found that miR-223-3p and KIF4A play important roles in regulating oxidative stress and apoptosis in breast cancer cells. Specifically, miR-223-3p promoted apoptosis by inhibiting the expression of KIF4A, increasing the accumulation level of ROS and MDA, and inhibiting the activity of SOD while KIF4A was overexpressed.

lncRNA MIR503HG inhibits cell proliferation and promotes apoptosis in TNBC cells via the miR-224-5p/HOXA9 axis

Background: Triple-negative breast cancer (TNBC) accounts for approximately 10 to 15% of all breast cancer cases. The management of TNBC cases will significantly improve once molecular mechanisms specific to TNBC cells will be identified and treated accordingly. Evidence suggests that TNBC cells display deregulated kinase-dependent signaling cascades that differ from non-triple-negative breast cancer cells. We hypothesized that uniquely divergent phospho-circuits could be distinguished between TNBC and non-TNBC cell lines. By revealing such unique, dysfunctional phospho-signaling network, our long-term objective is to identify kinases that underpin triple-negative breast cancer development, and can be inhibited using targeted therapy. Methods: The kinome activity of TNBC and non-TNBC cell lines was identified (HCC70, MDA-MB-231, MDA-MB-436 compared to AU565, MCF-7, T47D). The functional phospho-signature of each breast cancer cell was analyzed using a high throughput experimental platform that monitors the level of activity of myriad kinases at once. This technique uses 242 phospho-sensing probes and 78 controls in an aqueous-based assay to simultaneously and directly measure the phospho-catalytic activity of phosphorylating enzymes in cell lysates. We mapped the most significantly deranged phospho-signaling cascades and the related kinases. Results: Using 6 cell lines tested under various conditions, we generated 72 phospho-signatures, out of a total of 23,040 data points. After validating the repeatability and robustness of the assay, the kinase activity signature of each breast cancer cell line was analyzed and compared to each other using unsupervised hierarchical clustering. The phospho-sensing assay revealed the heterogeneity of kinase activity networks among breast cancer cells. These data also established that phospho-signaling cascades related to AKT, ERK, and SRC kinases were differentially altered in TNBC and non-TNBC cell lines. Conclusions: We successfully identified unique phospho-circuits of TNBC and non-TNBC cell lines. Our goal is now to test whether specific kinase inhibitors can efficiently kill or prevent the growth of TNBC cell lines in culture and animal models. We will expand our approach into a high-content, functional kinome-screening platform to characterize the phospho-fingerprint of breast cancer cells and tissues, and explore the druggable, kinase-dependent mechanisms critical to triple-negative breast tumors. Citation Format: Bo Pan, Miki Mori, Pei Rong Evelyn Lee, Marij Hartog, Qiang Sun, Laura van 't Veer, Jean-Philippe Coppe. Functional mapping of the oncogenic kinome activity of triple-negative breast cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-05-09.

Tumor microenvironment substantially influences the process of tumorigenesis. Extracellular acidification within the tumor microenvironment is an indicator of an aggressive cancer and a marker for poor patient outcome. In solid tumors, hypoxia leads to extracellular acidosis. Carbonic anhydrases (CA) are thought to regulate intracellular and extracellular pH (pHi and pHe, respectively). To explore the effect of CAs in breast cancer, we compared the expression and activity of two membrane bound CAs, CAIX and CAXII, between triple negative breast cancer cells (TNBCs) and luminal breast cancer cells (LBCs). We chose five different TNBC and LBC lines. Our data show that, among the TNBC lines, CAIX expression increased in three of the five lines: HBL100, SUM159, and the new UFH-001 cells under hypoxic condition. UFH-001 cells also showed strong constitutive expression. None of these TNBC lines expressed CAXII or estrogen receptor (ER). In LBC lines, four of the five lines constitutively expressed CAXII: T47D, MCF7, SKBR and SUM52 cells. CAXII expression was not hypoxia-dependent. Each of the five luminal lines expressed ER. We also examined CA expression in a tumor graft model. In tumors grown from cells derived from TNBC patients, we observed CAIX expression in four of six sample sets. In tumors derived from ER-positive LBC patients, all five expressed CAXII. We also used the 18O exchange method to assess CA activity. Two TNBC lines: UFH-001 and HBL100 cells showed that CAIX activity increased in hypoxic conditions which was blocked by an impermeant sulfonamide CA inhibitor (N3500). In the luminal lines, we detected CAXII activity in T47D and MCF7 cells that was also inhibited by N3500. Like CAXII protein expression in these cells, CAXII activity was not affected by hypoxia. We also evaluated the effect of pH on CA activity in TNBC and LBC lines. Both CAIX and CAXII showed increased activity in response to reduced pH, which is expected in a bicarbonate-based system. However, UFH-001 cells also exhibited a hypoxic-dependent increase in CAIX activity which is associated with increased protein expression. In conclusion, these observations demonstrate that CAIX expression is associated with the TNBC phenotype. Based on our activity data, we would predict that CA activity in TNBC tumors will be sensitive to both hypoxia (based on enhanced expression) and reduced pH. This change in activity may serve to regulate pH in the tumor microenvironment favoring an aggressive phenotype. On the other hand, LBC tumors, which are ER-positive, are only associated with CAXII expression. In luminal cells, we expect that only pH and not hypoxia will affect CAXII activity. This may, in part, explain the more positive prognosis in patients with CAXII expression. Note: This abstract was not presented at the meeting. Citation Format: Zhijuan Chen, Mam Y. Mboge, Chingkuang Tu, Lingbao Ai, Coy Heldermon, Susan C. Frost. Comparison of carbonic anhydrase & activity between triple-negative & luminal breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5936. doi:10.1158/1538-7445.AM2017-5936

Increasing evidence had suggested important roles of protein kinase C (PKC)-mediated phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) in modulating various cellular processes, including cancer cell growth and metastasis. However, there is a lack of information regarding to the roles of MARCKS and its pSer 159/163 phosphorylated product in breast cancer cells and tissues. To tackle this concern, we recently initiated both immunohistochemical and western blot analyses on a series of clinical specimens and several breast cancer cell lines. We have observed a significant elevation of phospho-MARCKS associated with advanced-stage breast cancer tissues as compared with benign ones, particularly in triple-negative breast cancer (TNBC) tissues and cell lines. Knockdown of MARCKS expression resulted in suppressions of lamellipodia/filopodia formation and migration/invasion as well as down-regulation of Src activity in these TNBC cell lines. Of note, MARCKS-silenced TNBC cells grew much more slowly and more sensitive to paclitaxel than the control and parental cells. Treatment with paclitaxel was shown to induce MARCKS phosphorylation in a dose-dependent manner, which may be related to an enhanced paclitaxel resistance in some of TNBC cells. However, PKC inhibitors were able to abrogate paclitaxel-induced MARCKS phosphorylation. Consistent with this potential, treatments of TNBC cells with a MARCKS N-terminus sequence peptide, namely MANS, to down regulate PKC/MARCKS pathway not only attenuated the associated aggressive phenotype but also synergistically enhanced paclitaxel-induced cytotoxicity in vitro and in vivo. These results demonstrate an association of PKC-mediated MARCKS phosphorylation with breast cancer malignancy potential and MARCKS phosphorylation as a predictor of paclitaxel resistance in TNBC cells. It is suggestive that an inhibition of PKC/MARCKS pathway may serve as an alternative therapeutic strategy for enhancing efficacy of chemotherapy. Citation Format: Ching-Hsien Chen, Muhammad Arif, Wen-Hsin Chang, Yuan Yuan, Jing Zhai, David K. Ann, Reen Wu. PKC/MARCKS pathway is a novel therapeutic target associated with breast cancer malignancy potential and paclitaxel resistance. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2769. doi:10.1158/1538-7445.AM2014-2769

Background: Triple-negative breast cancer (TNBC) accounts for 10-15% of all breast cancer cases. A major area of innovation in TNBC is identifying potential treatment targets, especially in TNBC cells which survive chemotherapy. Our previous study showed that TNBC cells displayed deregulated kinase-dependent signaling cascades, and uniquely divergent phospho-circuits could be distinguished between TNBC vs non-TNBC cell lines [2014 SABCS abstract 1672, poster P2-05-09]. We further hypothesized that specific dysfunctional phospho-signaling network played a key role in the early adaptive changes in DNA damage response of TNBC cells exposed to DNA damaging chemotherapeutic agents. Methods: TNBC cell lines MDA-MB-231 and MDA-MB-436 were treated with 5-fluorouracil (5-Fu), carboplatin and doxorubicin at their respective half maximal inhibitory concentrations (ic50s). MiSeq gene sequencing of the untreated vs treated TNBC cells was performed to investigate whether exposure to chemotherapy agents for 3-day's duration would induce additional adaptive genetic mutation. Apoptosis and cell-cycle distribution of the untreated and treated TNBC cells were analyzed with flow cytometry. The functional phospho-signature of each TNBC cell sample was analyzed using a high throughput experimental platform that monitors the level of activity of myriad kinases at once. This technique used over 450 phospho-sensing probes, including over 150 controls in an aqueous-based assay to simultaneously and directly measure the phospho-catalytic activity of phosphorylating enzymes in cell lysates. The kinome activities of the untreated vs treated TNBC cell lines were compared respectively, and the most significantly deranged and functionally altered phospho-signaling cascades and their related kinases were identified as the early adaptive changes of the survived TNBC cells after the 3-day exposure to DNA damage chemotherapies. Results: Using the two TNBC cell lines treated with the three chemotherapies, we made 8 cell line samples, including 6 treated and 2 untreated as the control. MiSeq gene sequencing showed no significant additional adaptive genetic mutations in the treated TNBC cells after the 3-day short-term exposure to 5-Fu, carboplatin and doxorubicin. 36 phospho-signatures were generated and validated for repeatability and robustness. The kinase activity signature of each TNBC sample was analyzed and compared to each other using unsupervised hierarchical clustering. The phospho-sensing assay revealed that phospho-signaling cascades related to CHK1/2 and IKK kinases were differentially altered in the untreated vs treated TNBC cell lines, which, when respectively inhibited by AZD7762 and IKK16, successfully increased growth inhibition and cell death of TNBCs. Conclusions: We identified specific phospho-fingerprints of the early adaption of TNBC cell lines and combinatorial targeted therapies that improve treatment outcome. Our next goal is to identify specific phosphorylation cascades in a broader range of cell lines and tumor tissues, to explore the actionable, kinase-dependent mechanisms critical to the DNA damage-induced adaptive reprogramming of TNBCs and early changes driving drug-resistance. Citation Format: Pan B, Olow A, Sun Q, Mori M, Lee PRE, Hartog M, Wang C, Wolf D, Yau C, van 't Veer L, Coppé J-P. Functional detection and inhibition of the targetable oncogenic kinome of chemotherapy-treated triple-negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-03.

Introduction. Triple negative breast cancer (TNBC), which comprises 15-20% of all breast cancers, is the deadliest and most aggressive subtype. TNBC affects a younger patient population and is associated with an increased risk of distant recurrence, higher rates of metastases, earlier time to recurrence, and worse prognosis after recurrence. AMP-activated protein kinase (AMPK) is a major cellular energy regulator that has recently been implicated in cancer progression. AMPK can act as a tumor suppressor by decreasing anabolism and inducing cell cycle arrest. FND 4b is a novel AMPK activator that has been shown to inhibit growth and induce apoptosis in colon cancer, but its effects have not been studied in breast cancer. The purpose of this project is to test the effects of FND 4b on AMPK activation, cellular proliferation, and apoptosis in several types of breast cancer—with a particular emphasis on TNBC. Methods. (i) To assess signaling pathways, estrogen-receptor positive (ER+) breast cancer cells (MCF-7 and T-47D), TNBC cells (MDA-MB-231 and HCC-1806), and breast cancer stem cells were treated with FND 4b (0, 1, 2.5, 5, 10, and 20 µM) for 24 h. Immunoblot analysis assessed phosphorylated and total forms of AMPK, acetyl CoA carboxylase (ACC), and ribosomal protein S6 as well as cyclin D1 and cleaved PARP. (ii) Growth assays with sulforhodamine B (SRB) were performed by treating ER+ breast cancer cells and TNBC cells with FND 4b (0, 2.5, 5, 10, and 20 µM) for 72 h and then quantifying cellular protein content. Proliferation was also assessed by treating all cell lines with FND 4b (0 or 5 µM) for 72 h and then counting viable cells. (iii) Cell death was assessed by treating ER+ breast cancer cells and TNBC cells with FND 4b (0, 2.5, 5, and 10 µM) for 72 h and then quantifying fragmented DNA in the cytoplasm by ELISA. Results. (i) FND 4b treatment for 24 h increased AMPK activation with concomitant decreases in ACC activity, phosphorylated S6, and cyclin D1 in ER+ breast cancer, TNBC, and breast cancer stem cells. (ii) FND 4b treatment for 72 h (5 µM) decreased proliferation in all breast cancer cells, while dose-dependent decreases in growth were also observed in ER+ breast cancer and TNBC cells. (iii) Increases in apoptosis were found in ER+ breast cancer and one TNBC cell line with FND 4b treatment for 72 h. Conclusions. Our findings indicate that FND 4b can decrease proliferation for a variety of breast cancers by activating AMPK—with notable effects on TNBC. The reductions in growth were mediated through decreases in fatty acid synthesis (ACC), mTOR signaling (S6), and cell cycle flux (cyclin D1). ER+ breast cancer cells were more susceptible to FND 4b-induced apoptosis, but MDA-MB-231 cells still underwent apoptosis with higher dose FND 4b treatment. Further development of FND compounds—with extensions to animal studies—could result in a novel therapeutic agent in the treatment of TNBC. Citation Format: Jeremy Johnson, Piotr Rychahou, Vitaliy M. Sviripa, Heidi L. Weiss, David Watt, B. Mark Evers. FND 4b decreases proliferation and increases apoptosis of triple negative breast cancer through AMPK activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3868.

Background: Breast cancer constitutes 30% of all new cancer cases in women. Despite steady decrease in breast cancer mortality, the lack of therapeutic targets is still a major problem. While the hormone-receptor positive (ER+) and human epidermal growth factor receptor 2-positive (HER2+) breast cancers respond to current targeted therapies, no targeted therapy is available for the treatment of triple negative breast cancer (TNBC), which lacks the expression of ER alpha (ER), progesterone receptor (PR), and HER2-receptor. Hence, there is an urgent need for effective targets against this sub-type of breast cancer. Previous studies in our laboratory used gene expression profiling of human breast cancers to identify kinases overexpressed in ER-negative breast cancers. One of these highly expressed kinases is the maternal embryonic leucine-zipper kinase (MELK). MELK is a serine/threonine protein kinase known to have a role in cell cycle progression, apoptosis and DNA repair. The purpose of this study was to test the hypothesis that MELK is required for the growth and migration of TNBC. Methods: RNA and protein was isolated from a panel of TNBC and ER-positive breast cancer cell lines and MELK expression was quantified by qPCR and immunoblotting. To determine whether MELK regulates cell growth, ER-positive and TNBC cell lines were transfected with siRNA targeting MELK and cell number was measured by manual counting. Anchorage-independent growth was measured using soft agar assays. Effect on migration and invasion was determined using Boyden Chamber assays. Immunostaining with actin-phalloidin was performed on MELK knockdown cells to determine effect of MELK loss on the cytoskeleton. Results: MELK mRNA and protein levels were significantly higher in TNBC cell lines compared to ER-positive breast cancers. Knockdown of MELK suppressed growth (≥50% growth inhibition) in six TNBC cell lines but had no effect on growth of six ER positive cell lines. Colony formation was also greatly reduced in TNBC cell lines but was not affected in ER positive cell lines upon siRNA knockdown of MELK. In addition, knockdown of MELK reduced migration of three TNBC cell lines (MDAMB231, MDAMB468 and Hcc70) but had no effect on the ER-positive cell line (MCF7). Decreased staining of actin filaments was observed in cell lines where migration was reduced upon MELK knockdown suggesting a role of MELK in formation of actin cytoskeleton. Current studies are focused on understanding how MELK regulates the cell growth and migration in TNBC. Conclusions: MELK is an important growth regulator of TNBC, but not of ER positive breast cancers. Our results indicate that MELK promotes cell migration in TNBC cells. These findings suggest that MELK is a promising target for the treatment of TNBC. Supported by a Susan G. Komen for the Cure Promise Grant (KG081694), and the John Charles Cain Award. Citation Format: Nidhi Batra, Corey Speers, Ivan Uray, Abhijit Mazumdar, Anna Tsimelzon, Susan Hilsenbeck, Gordon Mills, Powel Brown. Maternal embryonic leucine zipper kinase is critical for the growth and migration of triple negative breast cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3299. doi:10.1158/1538-7445.AM2014-3299

Background Triterpenoid saponins from sea cucumbers exhibit significant antitumour, antifungal, and antibacterial activities. However, the associated molecular mechanisms have yet to be elucidated. In this study, we screened and explored the antitumour activity and underlying mechanisms of triterpenoid saponins isolated from Thelenota ananas. Methods We isolated and purified sea cucumber saponins, determined their chemical structures, and confirmed their function in vitro. We also screened and explored the antitumour activity and underlying mechanisms of triterpenoid saponins isolated from Thelenota ananas. Results Four saponins were discovered from sea cucumber Thelenota ananas collected from the South China Sea. We found that stichloroside C2 (STC2) inhibited the proliferation and clonogenesis of the human triple-negative breast cancer (TNBC) cell line MDA-MB-231 and mouse TNBC cell line 4 T1 in a dose-dependent manner and induced apoptosis and cycle arrest in these two TNBC cell lines. STC2 induced DNA damage in two TNBC cell lines and significantly increased the protein expression level of the DNA double-strand break marker γ-H2AX. STC2 downregulated the protein expression levels of phosphorylated cyclin-dependent kinase 1 (CDK1), cyclin B1, CDK2, and cyclin A2 in MDA-MB-231 and 4 T1 cells. STC2 upregulated Bax and cleaved PARP protein expression in two types of breast cancer cells. In addition, STC2 promoted E-cadherin expression; inhibited vimentin expression; upregulated the phosphorylation levels of the mitogen-activated protein kinase (MAPK) signalling pathway-related proteins p38, JNK, and ERK1/2; and downregulated Akt phosphorylation. Conclusions STC2 exerts anti-TNBC activity, inhibits epithelial–mesenchymal transition (EMT), and induces apoptosis by regulating the cell cycle, EMT-related proteins, and MAPK signalling pathway.

The anticancer effects and mechanisms of marine sponge Aaptos suberitoides were rarely assessed, especially for methanol extract of A. suberitoides (MEAS) to breast cancer cells. This study evaluated the differential suppression effects of proliferation by MEAS between breast cancer and normal cells. MEAS demonstrated more antiproliferation impact on breast cancer cells than normal cells, indicating oxidative stress-dependent preferential antiproliferation effects on breast cancer cells but not for normal cells. Several oxidative stress-associated responses were highly induced by MEAS in breast cancer cells but not normal cells, including the generations of cellular and mitochondrial oxidative stress as well as the depletion of mitochondrial membrane potential. MEAS downregulated cellular antioxidants such as glutathione, partly contributing to the upregulation of oxidative stress in breast cancer cells. This preferential oxidative stress generation is accompanied by more DNA damage (γH2AX and 8-hydroxy-2-deoxyguanosine) in breast cancer cells than in normal cells. N-acetylcysteine reverted these MEAS-triggered responses. In conclusion, MEAS is a potential natural product for treating breast cancer cells with the characteristics of preferential antiproliferation function without cytotoxicity to normal cells in vitro.

Role of syndecan-4 in angiogenesis and vasculogenic mimicry in triple negative breast cancer cells.

BACKGROUND: CDK4/6 regulates the G1-S phase transition by phosphorylating the retinoblastoma protein (Rb). Given their potent clinical efficacy, CDK4/6 inhibitors used in combination with hormone receptor (HR) blockade (with an aromatase inhibitor or fulvestrant) are emerging as the standard of care for patients with metastatic HR-positive breast cancers. The CDK4/6 inhibitors palbociclib and ribociclib are FDA-approved for use in HR-positive breast cancer patients, and abemaciclib is currently in phase III trials. We observed that approximately 74% (25/34) of breast cancer cell lines had high phosphorylated Rb (phospho-Rb) expression levels and that triple-negative breast cancer (TNBC) cell lines often expressed phospho-Rb, suggesting that targeting phospho-Rb via CDK4/6 inhibition may be effective against TNBC. The histone deacetylase (HDAC) inhibitors increase p21Cip1 levels, promoting proteasomal degradation of cyclin B1 and resulting in G2/M arrest. Entinostat is an oral, class 1, selective HDAC inhibitor currently in phase III testing in HR-positive breast cancer. Preclinical and clinical data demonstrate that entinostat, in combination with HR blockade, has anticancer activity. Our group recently reported that entinostat combined with other anticancer drugs induced apoptosis via induction of proapoptotic proteins such as Noxa and Bim in breast cancer cell lines. Based on these findings, we hypothesized that entinostat-induced apoptosis and palbociclib-induced cell cycle arrest synergize to produce enhanced antitumor effects in estrogen receptor (ER)-positive breast cancer and TNBC cell lines with high phospho-Rb expression levels. METHODS: We assessed the combination antitumor effects and their mechanisms via CellTiter Blue and sulforhodamine B assays, flow cytometry, apoptosis (caspase 3/7) assays, anchorage-independent growth assays, Western blotting, reverse phase protein array (RPPA), and mammary fat pad xenograft mouse models. RESULTS: RPPA data showed that ER-positive and TNBC cell lines more often expressed phospho-Rb than did other breast cancer cell subtypes (7/10 and 8/17 cell lines, respectively). We found that the combination of entinostat and palbociclib synergistically inhibited tumor cell proliferation (combinational index less than 1.0), reduced in vitro colony formation (P < 0.05), inhibited in vivo tumor growth in ER-positive MCF-7 breast cancer cells (P < 0.05), and inhibited tumor growth in TNBC xenograft mouse models (MDA-MB-231) more effectively than did either drug alone. CONCLUSION: Taken together, our data provide evidence that combining entinostat with palbociclib enhances the antitumor effects of these drugs. Along with our continued effort to determine predictive biomarkers, our findings justify conducting a clinical trial of combination treatment with entinostat and palbociclib in patients with ER-positive breast cancer or TNBC. Citation Format: Lee J, Lim B, Pearson T, Tripathy D, Ordentlich P, Ueno NT. The synergistic antitumor activity of entinostat (MS-275) in combination with palbociclib (PD 0332991) in estrogen receptor-positive and triple-negative breast cancer [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 P5-21-15.

Objective To explore the expression of tiny RNA-25 (microRNA-25, miR-25) in the plasma、tissues of triple-negative breast cancer (TNBC) patients and cell lines, to investigate the potential molecular mechanisms of miR-25 on migration and invasion of TNBC. Methods Real-time fluorescent quantitative PCR was used to detect the expression of miR-25 in the plasma of TNBC patients. Linked omics web platform was used to analyse miR-25 level in samples of TNBC and non-TNBC. Real-time fluorescent quantitative PCR was also used to detect the miR-25 level in TNBC cell lines. The wound healing and transwell assay was applied to assess the effects on migration and invasion of TNBC cell lines which transfected with miR-25 inhibitor or the negative control. The luciferase reporter assay was used to validate the relationship between miR-25 and the sphingosine-1-phosphate phosphatase 1 (SGPP1) in HEK293T cell. The wound healing and transwell assay was used to detect the migration and invasion ability of TNBC cell lines when cotransfected with pCMV6-SGPP1 and miR-25. Furthermore, Western blot was performed to detect the SGPP1 level in TNBC cell lines. Results The expression of miR-25 was significantly elevated in the plasma of 86 TNBC patients compared with the healthy controls (P value was 0.031). LinkedOmics web platform analysis showed that miR-25 expression was significantly higher in TNBC samples than in non-TNBC samples with Luminal A or Luminal B (P value was<0.001 and 0.006). The level of miR-25 was also elevated in TNBC cell lines HS578T, HCC1806, MDA-MB-231 and BT549(P value was 0.006, 0.01, 0.029 and 0.046). The MDA-MB-231 and HS578T cells which transfected with miR-25 inhibitor exhibited a significant slower wound healing rate than control (P value was 0.035 and 0.001). At the same time, when transfected with miR-25 inhibitor, MDA-MB-231 and HS578T both exhibited a decreased invasion ability compared with the control group(P value was 0.002 and 0.001). LinkedOmics web platform analysis showed that sphingosine-1-phosphate phosphatase 1 (SGPP1) gene level was negatively correlated with miR-25 in the tissues of TNBC patients (P value was 0.037). The luciferase reporter assay validated that SGPP1 was a directed target of miR-25. The western blot assay indicated that the SGPP1 level was increased in MDA-MB-231 and HS578T after transfection with miR-25 inhibitor. Over-expression of SGPP1 could abrogate the positive effects of miR-25 on migration and invasion when pCMV6-SGPP1 was cotransfected with miR-25 (P value was all 0.002). Conclusions MiR-25 was elevated in both plasma and tissues of TNBC patients and also increased in TNBC cell lines. Transfection of MDA-MB-231 and HS578T cells with miR-25 inhibitor resulted in reduced migration and invasion. Moreover, SGPP1 was identified as a novel target of miR-25. The ability of miR-25 to promote TNBC cell migration and invasion is attributable to its effect on SGPP1 suppression. Key words: Triple negative breast neoplasms; microRNAs; Membrane proteins; Phosphoric monoester hydrolases; Neoplasm metastasis

Introduction: Diagnosis of triple negative breast cancer (TNBC) is associated with adverse prognosis particularly in case of chemotherapy resistance. TNBC is a heterogeneous entity and seems to consist of at least six distinct molecular subtypes (Lehman subtypes) with distinct chemotherapy sensitivity. The cytotoxic agent eribulin induces tumor cell apoptosis through depolymerization of the cell spindle apparatus. Based on clinical data it has recently been suggested that TNBC is particularly sensitive against eribulin. The goal of this analysis was to compare (i) TNBC vs. non TNBC lines and (ii) cell lines of distinct TNBC subtypes with regard to eribulin sensitivity in vitro. Methods: 17 established breast cancer cell lines comprising both TNBC (4 basal-like 1/2; 1 mesenchymal; 3 mesenchymal stem cell; 1 interleukin; 2 luminal AR; 1 unclassified) and non-TNBC (n=5) phenotypes were cultured and subjected to cell viability assay (MTT test), migration experiment (scratch assay), apoptosis analysis (Western Blot experiment for PARP cleavage) and quantitative RT-PCR analysis (for GABRP gene expression) after exposure to eribulin or control. Furthermore, gene expression of 8 genes known to induce malignant transformation (MMP7, ELF5, YBX1, RARRES1, PRNP, SOX 10, EGFR and GABRP) was analyzed via quantitative RT-PCR analysis in the triple negative cell line MDA-MB 231 after exposure to eribulin or control. Results: The effect of eribulin on the cell viability varied to a lesser extent among the TNBC compared to the non-TNBC cell lines though we could not observe a significant difference between both groups. Mentionable the TNBC cell line DU 4475 representing the interleukin phenotype displayed a significant stronger resistance to eribulin compared to all other phenotypes. A decelerated migration could be observed in the TNBC cell line MDA-MB 231 after exposure to the IC50 concentration of eribulin compared to non-treated cells. Induction of apoptosis by eribulin treatment was verified by PARP cleavage in various TNBC cell lines. GABRP known to be overexpressed especially in basal like TNBC showed a slight increase in gene expression after exposure to eribulin in various phenotypes of TNBC - most prominent in MDA-MB 231. Additionally, upregulation of ELF5 and downregulation of YBX1 and PRNP, and, to a lesser extent, of MMP7 and SOX 10 gene expression could be investigated in MDA-MB 231 after eribulin treatment. Conclusion: We did not observe a significant association with regard to eribulin sensitivity between TNBC and non-TNBC. Chemotherapy sensitivity varied to a lesser extent among TNBC cell lines compared to non-TNBC cell lines. Eribulin inhibits cell proliferation and migration, induces apoptosis in TNBC, and influences gene expression of overexpressed genes in TNBC known to participate in and induce malignant transformation. Though the current work did not explicitly specify one phenotype of TNBC for eribulin treatment regarding chemotherapy sensitivity, we identified possible target genes influenced by eribulin treatment, e. g. GABRP, and therefore need further investigation for a potential treatment approach combining eribulin with e. g. GABRP inhibitor. Citation Format: Bräutigam K, Mitzlaff K, Uebel L, Steinert G, Köster F, Polack S, Rody A, Liedtke C. Association between phenotype of triple negative breast cancer cell lines and sensitivity against eribulin mesylate in vitro. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-08-07.

Statin induces inhibition of triple negative breast cancer (TNBC) cells via PI3K pathway