Hs578T Cells Research Articles

Antitumoral Activity of a CDK9 PROTAC Compound in HER2-Positive Breast Cancer.

Cyclin-dependent kinases (CDKs) are a broad family of proteins involved in the cell cycle and transcriptional regulation. In this article, we explore the antitumoral activity of a novel proteolysis-targeting chimera (PROTAC) compound against CDK9. Breast cancer cell lines from different subtypes were used. Transcriptomic mapping of CDKs in breast cancer demonstrated that the expression of CDK9 predicted a detrimental outcome in basal-like tumors (HR = 1.51, CI = 1.08–2.11, p = 0.015) and, particularly, in the luminal B subtype with HER2+ expression (HR = 1.82, CI = 1.17–2.82, p = 0.0069). The novel CDK9 PROTAC, THAL-SNS-032, displayed a profound inhibitory activity in MCF7, T47D, and BT474 cells, with less effect in SKBR3, HCC1569, HCC1954, MDA-MB-231, HS578T, and BT549 cells. The three cell lines with HER2 overexpression and no presence of ER, SKBR3, HCC1569, and HCC1954 displayed an EC50 three times higher compared to ER-positive and dual ER/HER2-positive cell lines. BT474-derived trastuzumab-resistant cell lines displayed a particular sensitivity to THAL-SNS-032. Western blot analyses showed that THAL-SNS-032 caused a decrease in CDK9 levels in BT474, BT474-RH, and BT474-TDM1R cells, and a significant increase in apoptosis. Experiments in animals demonstrated an inverse therapeutic index of THAL-SNS-032, with doses in the nontherapeutic and toxic range. The identified toxicity was mainly due to an on-target off-tumor effect of the compound in the gastrointestinal epithelium. In summary, the potent and efficient antitumoral properties of the CDK9 PROTAC THAL-SNS-032 opens the possibility of using this type of compound in breast cancer only if specifically delivered to cancer cells, particularly in ER/HER2-positive and HER2-resistant tumors.

Density-Dependent Migration Characteristics of Cancer Cells Driven by Pseudopod Interaction.

The ability of cancer cells to invade neighboring tissue from primary tumors is an important determinant of metastatic behavior. Quantification of cell migration characteristics such as migration speed and persistence helps to understand the requirements for such invasiveness. One factor that may influence invasion is how local tumor cell density shapes cell migration characteristics, which we here investigate with a combined experimental and computational modeling approach. First, we generated and analyzed time-lapse imaging data on two aggressive Triple-Negative Breast Cancer (TNBC) cell lines, HCC38 and Hs578T, during 2D migration assays at various cell densities. HCC38 cells exhibited a counter-intuitive increase in speed and persistence with increasing density, whereas Hs578T did not exhibit such an increase. Moreover, HCC38 cells exhibited strong cluster formation with active pseudopod-driven migration, especially at low densities, whereas Hs578T cells maintained a dispersed positioning. In order to obtain a mechanistic understanding of the density-dependent cell migration characteristics and cluster formation, we developed realistic spatial simulations using a Cellular Potts Model (CPM) with an explicit description of pseudopod dynamics. Model analysis demonstrated that pseudopods exerting a pulling force on the cell and interacting via increased adhesion at pseudopod tips could explain the experimentally observed increase in speed and persistence with increasing density in HCC38 cells. Thus, the density-dependent migratory behavior could be an emergent property of single-cell characteristics without the need for additional mechanisms. This implies that pseudopod dynamics and interaction may play a role in the aggressive nature of cancers through mediating dispersal.

Abstract P5-10-03: Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome

Abstract TP53 is the most frequently mutated gene in most types of human cancer, including breast cancer. The triple-negative breast cancer (TNBC) subtype in particular displays TP53 mutation in approximately 80% of patients. Unlike other breast cancer subtypes (e.g., ER/PR-positive or HER2-positive), TNBC patients currently lack an approved highly effective targeted therapy. Notably, most TNBC acquires TP53 mutations, which, in addition to the loss of canonical p53 functions, can result in gain-of-function, activating different cellular mechanisms involved in tumor phenotypes such as proliferation, metastasis, invasiveness, and angiogenesis. Here we evaluate the role of mutant p53 in cancer phenotypes of TNBC cell lines, in particular their inflammatory profile. As expected, loss of p53 protein by small interfering RNA depletion did not show a major impact on the cell viability of MDA-MB231 and Hs578t cells in MTT assay. However, depleting mutant p53 knockdown made MDA-MB231 cells more susceptible to treatment with methyl-methane sulfonate, a genotoxic alkylating agent. Interestingly, cell invasion as measured by the transwell assay demonstrated that depletion of mutant p53 depletion decreased the invasiveness potential of MDA-MB231 and Hs578t cells which was substantiated with decreased migration of MDA-MB231 cells in a scratch assay over 24 h. RNA sequencing of MDA-MB231 and Hs578t cells revealed that mutant p53 knockdown decreased the expression of several constitutively expressed pro-inflammatory cytokines such as IL8, IL6, CXCL2, and CXCL3, but not genes associated with survival to alkylating agents (NRF2 and Endoplasmic reticulum stress markers) or genes typically regulated by wild-type p53 when compared to control-silenced and MMS-treated cells as indicated by Pathway Enrichment Analysis using the Enrich R and DAVID tools. These results were confirmed by ELISA quantification of IL8, IL6, and CXCL2 in MDA-MB231 and Hs578t transfected with two sequences of siRNA targeting mutant p53. On the other hand, RNA sequencing revealed some constitutively expressed genes known to be involved in breast cancer cells malignancy, such as PTGS2 (COX-2 enzyme gene) and MMP1, which were not affected by p53 knockdown. We found that MMP1, and PGE2 (the product of PTGS2/COX-2 enzyme), are upregulated by the ERK1/2 MAPK signaling pathway as determined in MDA-MB231 cells treated with the MEK1/2 inhibitor UO126 and sorafenib. UO126 and sorafenib also decreased IL8 and IL6 production. Furthermore, combined mutant p53 knockdown with MEK/ERK1/2 pathway inhibition caused a more pronounced IL8 and IL6 inhibition when compared to either p53 knockdown or UO126/sorafenib alone, whereas MMP1 and PGE2 levels were only reduced by MEK/ERK1/2 inhibitor treatments. Interestingly, neither mutant p53 knockdown impacted ERK1/2 phosphorylation status nor did UO126/sorafenib alter mutant p53 immunocontent. Reporter assays showed that mutant p53 promotes NFkappaB reporter activation, and MEK/ERK1/2 controls both NFkappaB and AP-1 transcription factors, both associated with the expression of the secretome components evaluated herein. Functional cell assays showed that concomitant inhibition of mutant p53 and MEK/ERK1/2 pathways reduce cell proliferation, invasion, and migration, indicating that mutant p53 protein gain of function cooperates with ERK1/2 MAPK signaling pathway to promote secretome production and malignant phenotypes in TNBC cell models. Citation Format: Raquel Nascimento das Neves, Aparna Gorthi, Alexander James Roy Bishop, Alfeu Zanotto Filho. Mutant p53 and ERK1/2 MAPK cooperate with the production of TNBC inflammatory secretome [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-10-03.

Abstract P5-17-10: Anti-progranulin (GP88) antibody AG01 inhibitory effect on the growth of triple negative breast cancer cells

Abstract Background: Triple negative breast cancer (TNBC) is characterized by invasiveness and short survival. Identifying novel TNBC targeted therapies to potentiate standard of care (SOC) therapy, is an unmet need. Progranulin (PGRN/GP88) is a biological driver of tumorigenesis, survival, and drug resistance in several cancers including breast cancer (BC). PGRN/GP88 tissue expression is an independent prognostic factor of recurrence while elevated serum PGRN/GP88 level is associated with poor outcomes such as progression of disease and shortened survival. Since PGRN/GP88 expression is elevated in 30% TNBC, we investigated the effect of inhibiting PGRN/GP88 effect on the proliferation and tumor growth of triple negative breast cancer cells. Methods: For this purpose, we have developed a neutralizing anti-human PGRN/GP88 monoclonal antibody AG01 and examined its effect on the proliferation, migration, signaling pathway activation and biomarker expression of two TNBC cell lines MDA-MB-231 and HS578-T expressing PGRN/GP88, both in vitro and in vivo. Results: The inhibition of PGRN/GP88 action by AG01 treatment reduced proliferation and migration in a dose-and time-dependent fashion in MDA-MB-231 and HS578-T cells. Western blot analysis showed decreased expression of phosphorylated protein kinases p-Src, p-AKT and p-ERK involved in proliferation and survival upon AG01 treatment for both cell lines. Transwell assay showed that AG01 treatment inhibited migration and invasion in a dose-dependent fashion. Microarray analysis of several oncoproteins in cells treated with AG01 or control antibody demonstrated the inhibition of the expression of several markers of migration and angiogenesis. In vivo xenograft studies with MDA-MB-231 cells injected in athymic nude mice showed that AG01 treatment inhibited tumor growth as well as Ki67 expression, mitotic index and microvessel counts when compared to antibody control treated mice. In vivo dose response of AG01 in MDA-MB-231 tumor bearing mice will be provided. Conclusion: TNBC is a disease with poor prognosis in need of novel targeted therapeutic solutions. PGRN/GP88 represents a therapeutic target for TNBC with two companion diagnostics (tissue test and ELISA to measure GP88 circulating levels). Blocking PGRN/GP88 with AG01 antibody treatment will provide novel targeted therapeutic option for TNBC which could address the issue of toxicity, and unresponsiveness associated with SOC. This work is supported by a grant CA 224718 from the National Cancer Institute to GS. Citation Format: Ginette Serrero, Rupa Guha, Jianping Dong, Binbin Yue. Anti-progranulin (GP88) antibody AG01 inhibitory effect on the growth of triple negative breast cancer cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-10.

Abstract PD3-01: Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer

Abstract Our previous findings have shown that increased inducible nitric oxide synthase (iNOS) expression is a poor prognostic indicator and associated with worse overall survival in TNBC patients. Growing evidence has also suggested that hyperactivation of the phosphoinositide-3-kinase (PI3K) survival signaling pathway is one of the most common oncogenic aberrations in TNBC. Nitric oxide (NO) is a unique molecule in its ability to target multiple oncogenic pathways in a spatial and temporal manner, such as PI3K, extracellular signal-regulated kinase (ERK), β-catenin pathway, transforming growth factor beta (TGFβ) signaling, and hypoxia-inducible factor (HIF). This study investigates the role of NO in PI3K-Akt pathway activation in human TNBC cells, in conditions with extracellular NO donor exposure and increased intracellular iNOS (NOS2) expression. TNBC cell lines BT549, HCC1937, Hs578T, SUM159, MDA-MB-231, and BT-20 were exposed to the NO donor DETA NONOate at increasing concentrations (0-500 µM) for 12 hours. Significant elevation in phosphorylation of Akt (Ser473 and Thr308) in response to increase NO donor concentration was found only in cell lines with wild-type PTEN and PIK3R1, such as SUM159, MDA-MB-231, and BT20. To evaluate whether NO-induced activation of Akt was PI3K-dependent, SUM159 (wild-type PTEN) and HCC1937 (PTEN mutant) cells were also pre-treated with PI3K inhibitor alpelisib before NO exposure. PI3K inhibition was only able to eliminate basal phospho-Akt and prevent NO-induced Akt activation in the SUM159 cell line. Furthermore, compared to vehicle control treated cells, we found that NO donor-induced activation enhanced phosphorylation in 15/18 Akt signaling proteins in SUM159 cells (PTEN intact) and 3/18 Akt signaling proteins in HCC1937 cells (PTEN loss). In Hs578T cells in which NOS2 was either overexpressed or knocked down via lentiviral transduction, we found that enhanced expression of NOS2 was associated with increased p-Akt expression and knock-down of NOS2 led to reduced p-Akt. This result suggests that iNOS, as opposed to other NOS isoforms, is more associated with modulating PI3K signaling in TNBC.We also analyzed TNBC data from The Cancer Genome Atlas and found that patients with positive iNOS mRNA expression had significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.0001] expression compared to patients with no iNOS expression. When we stratified this patient cohort based on both iNOS and PTEN expression, we only found significantly higher phospho-Akt (Ser473 and Thr308) [p≤0.01] expression in patients with positive iNOS and PTEN expression, supporting the hypothesis that NO may impair PTEN’s function as a PI3K antagonist. Ongoing studies are directed to investigate mechanisms of iNOS-PTEN physical interactions, iNOS-dependent post-translational modifications to PTEN, and whether iNOS and PTEN are dual prognostic markers in TNBC patients. Citation Format: Tejaswini P. Reddy, Liliana Guzman-Rojas, Roberto R. Rosato, Wei Qian, Hong Zhao, Jenny C. Chang. Inducible nitric oxide synthase activates PI3K/Akt signaling via PTEN S-nitrosylation in triple-negative breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD3-01.

Abstract P3-05-07: The response of histone deacetylase inhibitors in triple negative breast cancer

Abstract Breast cancer (BC) is the most prevalent type of cancer among women with triple negative breast cancer (TNBC) accounting for 15% of BC. The majority of BCs are hormone sensitive, TNBC, a more aggressive subtype is characterized by its negative expression of ER and PR, and lack of Her2/NEU amplification. Due to its receptor status, TNBC lacks targeted therapy. FK228 (Romidepsin), a class I and II histone deacetylase inhibitor (HDACi) has been approved by the FDA for the treatment of PTCL (peripheral T-cell lymphoma) and is a promising drug for TNBC treatment. FK228 mechanism of action was through cell cycle arrest and apoptosis (1,2). FK228 specifically targets the enzymes HDACs 1 and 2, tumor suppressor and other proteins thereby exerting epigenetic changes on cancer cells resulting in anti-tumor activity. To assess the role of HDACi, TNBC cell lines were treated with FK228 and Panobinostat (LBH, a panHDACi) and analyzed for suppression of cell cycle genes. Similar to PTCL, TNBC cell lines showed an increase in cell cycle arrest genes such as p21 and others subsequent to treatment with both drugs. Using PCR and crystal violet we identified changes in morphology and migration in 2k1, MDA-MB 231 and HS-578t cell lines treated with FK228 under in-vitro conditions. Further studies show a reversal of the epithelial to mesenchymal transition (EMT) in FK228 treated cells; specifically in key EMT genes CDH1 and ZEB2, both directly linked to HDAC1/2 activity. Similar results were replicated in 3D culture mammospheres. We are also further studying the effects of HDACi in vivo using PDXs (patient derived xenografts) in mouse models; the PDX is either injected as cells or implanted directly in the mammary pad. The PDX model is an excellent translational tool used to monitor growth patterns and recurrence, study metastasis and drug response. Thus far, the TU-BcX-2O0 PDX in vivo model demonstrated growth suppression in tumor growth with FK228 treatment. We are also studying FK228 in models TU-BX-4IC, 4M4 and 4QX. Metastasis in the models is monitored by H&E staining lung and liver tissue for further staining and analysis; we are also using IVIS imaging in an in vivo model consisting of GFP/luciferase transfected breast cancer cells MDA-MB-231 and SUM-159 to monitor both the proliferation and spread of disease in mice. Ultimately we aim to characterize the pathways altered by FK228 and the involvement of HDACs in tumorigenesis, metastasis and resistance within TNBC.

Subcellular regulation of glucose metabolism through multienzyme glucosome assemblies by EGF–ERK1/2 signaling pathways

A multienzyme metabolic assembly for human glucose metabolism, namely the glucosome, has been previously demonstrated to partition glucose flux between glycolysis and building block biosynthesis in an assembly size-dependent manner. Among three different sizes of glucosome assemblies, we have shown that large-sized glucosomes are functionally associated with the promotion of serine biosynthesis in the presence of epidermal growth factor (EGF). However, due to multifunctional roles of EGF in signaling pathways, it is unclear which EGF-mediated signaling pathways promote these large glucosome assemblies in cancer cells. In this study, we used Luminex multiplexing assays and high-content single-cell imaging to demonstrate that EGF triggers temporal activation of extracellular signal-regulated kinases 1/2 (ERK1/2) in Hs578T cells. Subsequently, we found that treatments with a pharmacological inhibitor of ERK1/2, SCH772984, or short-hairpin RNAs targeting ERK1/2 promote the dissociation of large-sized assemblies to medium-sized assemblies in Hs578T cells. In addition, our Western blot analyses revealed that EGF treatment does not increase the expression levels of enzymes that are involved in both glucose metabolism and serine biosynthesis. The observed spatial transition of glucosome assemblies between large and medium sizes appears to be mediated by the degree of dynamic partitioning of glucosome enzymes without changing their expression levels. Collectively, our study demonstrates that EGF–ERK1/2 signaling pathways play an important role in the upregulation of large-sized glucosomes in cancer cells, thus functionally governing the promotion of glycolysis-derived serine biosynthesis.

SLFN12 Over-expression Sensitizes Triple Negative Breast Cancer Cells to Chemotherapy Drugs and Radiotherapy.

Schlafen 12 (SLFN12) expression correlates with survival in triple negative breast cancer (TNBC). SLFN12 slows TNBC proliferation and induces TNBC differentiation, but whether SLFN12 affects the tumoral response to chemotherapy or radiation is unknown. We over-expressed SLFN12 in MDA-MB-231 cells using two different lentiviral vectors. We assessed viable cell numbers via crystal violet assay after treatment with carboplatin, paclitaxel, olaparib, zoledronic acid, camptothecin, or cesium irradiation. CHK1 and CHK2 phosphorylation was assessed by western blot and the effects of inhibiting CHK1/CHK2 by AZD7762 were examined. Key findings were confirmed in Hs578t and BT549 TNBC cells after adenoviral SLFN12 over-expression. SLFN12 over-expression increased TNBC sensitivity to radiation, carboplatin, paclitaxel, zoledronic acid, and camptothecin, but not to olaparib. SLFN12 over-expression decreased CHK1 and CHK2 phosphorylation after treatment with the DNA damaging agent camptothecin (CPT). The CHK1/CHK2 inhibitor diminished the significant cytotoxicity difference between over-expression and baseline SLFN12 levels in response to carboplatin. SLFN12 increases TNBC sensitivity to DNA-damaging agents at least in part by reducing CHK1/2 phosphorylation. This may contribute to improved survival in patients whose TNBC over-expresses SLFN12. Therefore, SLFN12 levels may be used to customize or predict radiotherapy and chemotherapy effects in TNBC.

CXCL8 is a prognostic biomarker and correlated with TNBC brain metastasis and immune infiltration

Patients with TNBC are associated with an increased risk of developing brain metastasis and shortest median survival post-brain metastasis-diagnosis. However, the regulatory mechanism of TNBC brain metastasis has not been addressed. Here, by a series of integrated analyses of differential gene expression profile from brain metastases and primary triple negative breast cancer, we identified 15 differentially expressed genes in both TNBC brain metastasis tissue samples and TNBC brain metastasis cell line. After analyzing the prognostic value of those 15 differentially expressed genes, we found that CXCL8 was the only gene associated with multiple prognostic indicators in both all-breast cancer and TNBC populations. Functional and pathway enrichment analyses demonstrated that CXCL8 was associated with humoral immune response and immune cell infiltration. CXCL8 expression had a positive correlation with three immune-related scores (ImmuneScore, ESTIMATEScore and StromalScore), and multiple types of immune cell infiltration, including macrophages, neutrophils and Th1 cells. Besides, we also verified the prometastatic effect of CXCL8, by treating MDA-MB-231 and Hs578t cells with different concentrations of recombinant human CXCL8. Taken together, our results suggest that CXCL8 can be used as a prognostic biomarker and is associated with TNBC brain metastasis and immune infiltration. Our findings provide a new perspective on TNBC brain metastasis and illustrate great potential to develop new CXCL8-targeted therapy for clinical TNBC patients.

Synergism of a novel MCL‑1 downregulator, acriflavine, with navitoclax (ABT‑263) in triple‑negative breast cancer, lung adenocarcinoma and glioblastoma multiforme.

Myeloid cell leukemia sequence 1 (MCL-1), an anti-apoptotic B-cell lymphoma 2 (BCL-2) family molecule frequently amplified in various human cancer cells, is known to be critical for cancer cell survival. MCL-1 has been recognized as a target molecule for cancer treatment. While various agents have emerged as potential MCL-1 blockers, the present study presented acriflavine (ACF) as a novel MCL-1 inhibitor in triple-negative breast cancer (TNBC). Further evaluation of its treatment potential on lung adenocarcinoma and glioblastoma multiforme (GBM) was also investigated. The anticancer effect of ACF on TNBC cells was demonstrated when MDA-MB-231 and HS578T cells were treated with ACF. ACF significantly induced typical intrinsic apoptosis in TNBCs in a dose- and time-dependent manner via MCL-1 downregulation. MCL-1 downregulation by ACF treatment was revealed at each phase of protein expression. Initially, transcriptional regulation via reverse transcription-quantitative PCR was validated. Then, post-translational regulation was explained by utilizing an inhibitor against protein biosynthesis and proteasome. Lastly, immunoprecipitation of ubiquitinated MCL-1 confirmed the post-translational downregulation of MCL-1. In addition, the synergistic treatment efficacy of ACF with the well-known MCL-1 inhibitor ABT-263 against the TNBC cells was explored [combination index (CI)<1]. Conjointly, the anticancer effect of ACF was assessed in GBM (U87, U251 and U343), and lung cancer (A549 and NCI-H69) cell lines as well, using immunoblotting, cytotoxicity assay and FACS. The effect of the combination treatment using ACF and ABT-263 was estimated in GBM (U87, U343 and U251), and non-small cell lung cancer (A549) cells likewise. The present study suggested a novel MCL-1 inhibitory function of ACF and the synergistic antitumor effect with ABT-263.

The Preliminary Study on the Proapoptotic Effect of Reduced Graphene Oxide in Breast Cancer Cell Lines.

Breast cancer is the most common cancer diagnosed in women, however traditional therapies have several side effects. This has led to an urgent need to explore novel drug approaches to treatment strategies such as graphene-based nanomaterials such as reduced graphene oxide (rGO). It was noticed as a potential drug due to its target selectivity, easy functionalisation, chemisensitisation, and high drug-loading capacity. rGO is widely used in many fields, including biological and biomedical, due to its unique physicochemical properties. However, the possible mechanisms of rGO toxicity remain unclear. In this paper, we present findings on the cytotoxic and antiproliferative effects of rGO and its ability to induce oxidative stress and apoptosis of breast cancer cell lines. We indicate that rGO induced time- and dose-dependent cytotoxicity in MDA-MB-231 and ZR-75-1 cell lines, but not in T-47D, MCF-7, Hs 578T cell lines. In rGO-treated MDA-MB-231 and ZR-75-1 cell lines, we noticed increased induction of apoptosis and necrosis. In addition, rGO has been found to cause oxidative stress, reduce proliferation, and induce structural changes in breast cancer cells. Taken together, these studies provide new insight into the mechanism of oxidative stress and apoptosis in breast cancer cells.

A curcumin analog CA-5f inhibits urokinase-type plasminogen activator and invasive phenotype of triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is one of the most aggressive types of breast cancer with poor outcomes. Patients with TNBC cannot benefit from targeted therapies such as Tamoxifen and Herceptin. The aim of the present study was to seek a preventive or therapeutic agent with a potential inhibitory effect on aggressive progression of TNBC. Anticancer effect of a natural compound curcumin have been demonstrated, however, development of more effective curcumin analogs with better bioavailability is needed. We investigated if a curcumin analog CA-5f could inhibit the invasive phenotype of TNBC cell lines in the present study. Treatment with CA-5f inhibited the viability of MDA‑MB‑231 and Hs578T TNBC cells, possible by inducing apoptosis. The invasive phenotypes of these cells were inhibited by CA-5f in a concentration-dependent manner. Protein expression of urokinase-type plasminogen activator (uPA), a serine protease known to degrade the extracellular matrix and lead to invasion, was markedly decreased by CA-5f in Hs578T cells. However, mRNA level of uPA was not altered by CA-5f, implicating that the effect of CA-5f was not through transcriptional regulation. Of note, CA-5f upregulated plasminogen activator inhibitor type (PAI)-1, which is known to inhibit uPA by interacting with urokinase-type plasminogen receptor, in TNBC cells. Taken together, these results demonstrated that CA-5f significantly inhibited the invasive phenotype of TNBC cells, possibly by decreasing the protein level of uPA through upregulating PAI-1. Our results may provide useful information on developing CA-5f as a potential therapeutic agent against malignant progression of TNBC.

Controlled Release of Doxorubicin for Targeted Chemo-Photothermal Therapy in Breast Cancer HS578T Cells Using Albumin Modified Hybrid Nanocarriers.

Hybrid nanomaterials have attracted research interest owing to their intriguing properties, which may offer new diagnostic options with triggering features, able to realize a new kind of tunable nanotherapeutics. Hybrid silica/melanin nanoparticles (NPs) containing silver seeds (Me-laSil_Ag-HSA NPs) disclosed relevant photoacoustic contrast for molecular imaging. In this study we explored therapeutic function in the same nanoplatform. For this purpose, MelaSil_Ag-HSA were loaded with doxorubicin (DOX) (MelaSil_Ag-HSA@DOX) and tested to assess the efficiency of drug delivery combined with concurrent photothermal treatment. The excellent photothermal properties allowed enhanced cytotoxic activity at significantly lower doses than neat chemotherapeutic treatment. The results revealed that MelaSil_Ag-HSA@DOX is a promising platform for an integrated photothermal (PT) chemotherapy approach, reducing the efficacy concentration of the DOX and, thus, potentially limiting the several adverse side effects of the drug in in vivo treatments.

PDGFRα Enhanced Infection of Breast Cancer Cells with Human Cytomegalovirus but Infection of Fibroblasts Increased Prometastatic Inflammation Involving Lysophosphatidate Signaling.

Human cytomegalovirus (HCMV) infects 40–70% of adults in developed countries. HCMV proteins and DNA are detected in tumors and metastases, suggesting an association with increased invasion. We investigated HCMV infection in human breast cancer cell lines compared to fibroblasts, a component of tumors, and the role of platelet-derived growth factor receptor-α (PDGFRα). HCMV productively infected HEL299 fibroblasts and, to a lesser extent, Hs578T breast cancer cells. Infection of another triple-negative cell line, MDA-MB-231, and also MCF-7 cells, was extremely low. These disparate infection rates correlated with expression of PDGFRA, which facilitates HCMV uptake. Increasing PDGFRA expression in T-47D breast cancer and BCPAP thyroid cancer cells markedly increased HCMV infection. Conversely, HCMV infection decreased PDGFRA expression, potentially attenuating signaling through this receptor. HCMV infection of fibroblasts promoted the secretion of proinflammatory factors, whereas an overall decreased secretion of inflammatory factors was observed in infected Hs578T cells. We conclude that HCMV infection in tumors will preferentially target tumor-associated fibroblasts and breast cancer cells expressing PDGFRα. HCMV infection in the tumor microenvironment, rather than cancer cells, will increase the inflammatory milieu that could enhance metastasis involving lysophosphatidate.

MicroRNA-495/TGF-β/FOXC1 axis regulates multidrug resistance in metaplastic breast cancer cells

Triple-negative metaplastic breast carcinoma (MBC) poses a significant treatment challenge due to lack of targeted therapies and chemotherapy resistance. We isolated a novel MBC cell line, BAS, which showed a molecular and phenotypic profile different from the only other metaplastic cell model, HS578T cells. To gain insight behind chemotherapeutic resistance, we generated doxorubicin (HS-DOX, BAS-DOX) and paclitaxel (HS-TX, BAS-TX) resistant derivatives of both cell lines. Drug sensitivity assays indicated a truly multidrug resistant (MDR) phenotype. Both BAS-DOX and BAS-TX showed up-regulation of FOXC1 and its experimental down-regulation re-sensitized cells to doxorubicin and paclitaxel. Experimental modulation of FOXC1 expression in MCF-7 and MDA-MB-231 cells corroborated its role in MDR. Genome-wide expression analyses identified gene expression signatures characterized by up-regulation of TGFB2, which encodes cytokine TGF-β2, in both BAS-DOX and BAS-TX cells. Pharmacological inhibition of the TGF-β pathway with galunisertib led to down-regulation of FOXC1 and increase in drug sensitivity in both BAS-DOX and BAS-TX cells. MicroRNA (miR) expression analyses identified high endogenous miR-495-3p levels in BAS cells that were downregulated in both BAS MDR cells. Transient expression of miR-495-3p mimic in BAS-DOX and BAS-TX cells caused downregulation of TGFB2 and FOXC1 and re-sensitized cells to doxorubicin and paclitaxel, whereas miR-495-3p inhibition in BAS cells led to increase in resistance to both drugs and up-regulation of TGFB2 and FOXC1. Together, these data suggest interplay between miR-495-3p, TGF-β2 and FOXC1 regulating MDR in MBC and open the exploration of novel therapeutic strategies.

Lentiviral CRISPR-guided RNA library screening identified Adam17 as an upstream negative regulator of Procr in mammary epithelium

BackgroundProtein C receptor (Procr) has recently been shown to mark resident adult stem cells in the mammary gland, vascular system, and pancreatic islets. More so, high Procr expression was also detected and used as indicator for subsets of triple-negative breast cancers (TNBCs). Previous study has revealed Procr as a target of Wnt/β-catenin signaling; however, direct upstream regulatory mechanism of Procr remains unknown. To comprehend the molecular role of Procr during physiology and pathology, elucidating the upstream effectors of Procr is necessary. Here, we provide a system for screening negative regulators of Procr, which could be adapted for broad molecular analysis on membrane proteins.ResultsWe established a screening system which combines CRISPR-Cas9 guided gene disruption with fluorescence activated cell sorting technique (FACS). CommaDβ (murine epithelial cells line) was used for the initial Procr upstream effector screening using lentiviral CRISPR-gRNA library. Shortlisted genes were further validated through individual lentiviral gRNA infection followed by Procr expression evaluation. Adam17 was identified as a specific negative inhibitor of Procr expression. In addition, MDA-MB-231 cells and Hs578T cells (human breast cancer cell lines) were used to verify the conserved regulation of ADAM17 over PROCR expression.ConclusionWe established an efficient CRISPR-Cas9/FACS screening system, which identifies the regulators of membrane proteins. Through this system, we identified Adam17 as the negative regulator of Procr membrane expression both in mammary epithelial cells and breast cancer cells.

Bioactive Abietane-Type Diterpenoid Glycosides from Leaves of Clerodendrum infortunatum (Lamiaceae).

In this study, two previously undescribed diterpenoids, (5R,10S,16R)-11,16,19-trihydroxy-12-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-17(15→16),18(4→3)-diabeo-3,8,11,13-abietatetraene-7-one (1) and (5R,10S,16R)-11,16-dihydroxy-12-O-β-d-glucopyranosyl-(1→2)-β-d-glucopyranosyl-17(15→16),18(4→3)-diabeo-4-carboxy-3,8,11,13-abietatetraene-7-one (2), and one known compound, the C13-nor-isoprenoid glycoside byzantionoside B (3), were isolated from the leaves of Clerodendrum infortunatum L. (Lamiaceae). Structures were established based on spectroscopic and spectrometric data and by comparison with literature data. The three terpenoids, along with five phenylpropanoids: 6′-O-caffeoyl-12-glucopyranosyloxyjasmonic acid (4), jionoside C (5), jionoside D (6), brachynoside (7), and incanoside C (8), previously isolated from the same source, were tested for their in vitro antidiabetic (α-amylase and α-glucosidase), anticancer (Hs578T and MDA-MB-231), and anticholinesterase activities. In an in vitro test against carbohydrate digestion enzymes, compound 6 showed the most potent effect against mammalian α-amylase (IC50 3.4 ± 0.2 μM) compared to the reference standard acarbose (IC50 5.9 ± 0.1 μM). As yeast α-glucosidase inhibitors, compounds 1, 2, 5, and 6 displayed moderate inhibitory activities, ranging from 24.6 to 96.0 μM, compared to acarbose (IC50 665 ± 42 μM). All of the tested compounds demonstrated negligible anticholinesterase effects. In an anticancer test, compounds 3 and 5 exhibited moderate antiproliferative properties with IC50 of 94.7 ± 1.3 and 85.3 ± 2.4 μM, respectively, against Hs578T cell, while the rest of the compounds did not show significant activity (IC50 > 100 μM).

Extracellular Vesicles Carry lncRNA SNHG16 to Promote Metastasis of Breast Cancer Cells via the miR-892b/PPAPDC1A Axis.

Breast cancer (BC) represents the most commonly diagnosed malignancy among women. Long non-coding RNAs (lncRNAs) can be transferred by extracellular vesicles (EVs) to participate in BC progression. This study demonstrated that SNHG16 expression was significantly increased in BC tissues and cells. Overexpression of SNHG16 promoted the migration, invasion, and epithelial–mesenchymal transition (EMT) of BC cells. SNHG16 was carried by EVs. Bioinformatics analysis predicted that SNHG16 regulated PPAPDC1A expression by sponging miR-892b, which was confirmed by RNA-fluorescence in situ hybridization (FISH), RT-qPCR, dual-luciferase gene reporter assay, and RNA immunoprecipitation (RIP). MDA-MB-157 and HS578T cells were transfected with pcDNA3.1-SNHG16, miR-892b-mimic, or si-PPAPDC1A for functional rescue experiments in vitro, and the cells were treated with MDA-MB-231 cell-derived EVs. The results confirmed that enhanced miR-892b expression partially eliminated the increase of migration, invasion, and EMT of BC cells mediated by SNHG16 or EVs. The lung metastasis model in nude mice was established by injecting HS578T cells via tail vein. The results showed that si-SNHG16 reduced the metastatic nodules and decreased the vimentin expression. In conclusion, EVs derived from BC cells transferred SNHG16 via the miR-892b/PPAPDC1A axis, thus promoting EMT, migration, and invasion of BC.

Functional and Biological Implications of CUX1 Mutations and Deletions in Myeloid Neoplasms

',“ authors equally contributeThe CUX1 gene, located on 7q22.1, has been characterized genetically as a haplo-insufficient tumor suppressor gene. Somatic CUX1 mutations (CUX1MT) and deletions (CUX1DEL) have been reported in myeloid neoplasms.The p200CUX1 is the most abundant isoform, containing 4 DNA-binding domains (CR1, CR2, CR3, and HD) with rapid “on” and “off” DNA-binding kinetics. Depending on promoter context, the p110 isoform can be either a repressor or an activator. We aim to define the clinical and biological/functional relevance of CUX1 lesions in myeloid neoplasms.We first established the clinical prevalence and phenotypic associations of CUX1 . In a cohort of 1480 patients with myeloid neoplasms including, lower-risk MDS (24%, n=359), higher-risk MDS (17%, n=249), pAML (22%, n=322), sAML (14%, n=205), MDS/MPN (15%, n=217) and MPN (9%, n=128), we correlated CUX1 lesions with clinical parameters, cytogenetic abnormalities, and molecular features including clonal architecture and concomitant somatic mutations. We found that somatic CUX1MT and CUX1DEL were detected in 4% and 6% of our cohort, respectively. All CUX1MT were heterozygous except in 6% were homozygous (uniparental disomy; UPD) and 7%, compounded heterozygous fashion. CUX1 is under expressed in 60% of MDS with -7/del(7q) and 70% of AML with -7/del(7q) vs. 18% and 8% of diploid controls (P=.004 and P <.0001,respectively). In addition, 15% of MDS and 5% of AML cases with cytogenetic abnormality other than -7/del(7q) showed low expression of CUX1 . Truncations and missense mutations were present in 25% and 75% of mutant cases, respectively. CUX1MT were more common in MDS (52%) than AML(16%) and MDS/MPN (32%; P=.02), while CUX1DEL more frequently exist in RAEB1/2 and sAML (64%) compared to lower-risk MDS (12%), pAML (12%) and MDS/MPN (14%; P <.0001). Regards concomitant genetic events, CUX1 lesions were significantly associated with TET2, ASXL1 and BCOR (P=.02, P=.004, P=.0009 respectively) compared to CUX1 wild type (WT). In contrast to WT which was mutually exclusive with FLT3 (3%). Clonal architecture analyses showed that 36% of CUX1 mutations were founder events with secondary BCOR (25%) and ASXL1 (17%) , while CUX1MT were subclonal in 64% in which TET2 (22%), BCOR (9%), and SF3B1 ( 9%) served as ancestral events. Prognostic analyses showed that both CUX1MT and CUX1DEL were significantly associated with shorter overall survival compared WT (CUX1MT: 56 vs. 94 mo.; P=.02; CUX1DEL: 46 vs. 94 mo., P=.004) where truncating mutations had a significant impact on worse prognosis than WT (39 v s. 94 mo.; P=.01).CUX1 functions as an auxiliary factor in base excision repair. Specifically, the CUT domains within CUX1 stimulate the enzymatic activities of the 8-oxoguanine DNA Glycosylase-1 (OGG1). We therefore verified whether genetic lesions of CUX1, either CUX1MTor CUX1DEL, delay oxidative DNA damage repair and thereby contribute to leukemogenesis by establishing a clonal mutator phenotype. First, we compared 8-oxoG cleavage activity in extracts from HCC1419 cells harboring CUX1 loss-of-heterozygosity (CUX1LOH), Hs578T cells diploid for CUX1 and Hs578T cells expressing CUX1 shRNA. We observed decreased 8-oxoG cleavage in HCC1419 cells and Hs578T-shCUX1 cells in which CUX1 expression was reduced. Secondly, we obtained bone marrow cells from healthy donors and patients with frameshift CUX1MTor del7q, and performed single cell gel electrophoresis (comet assays) to monitor DNA repair following exposure to H2O2. We found that repair of oxidized bases in genomic DNA was delayed in both samples with heterozygous and homozygous CUX1 inactivating mutations and in samples with CUX1DEL. Having noted that CUX1 lesions cause a defect in BER, we performed whole exome sequencing of CUX1MT (n=3) and WT (n=117). Strikingly, samples with either CUX1MT or low CUX1 expression (n=23) harbored a significantly higher number of mutations compared to WT and high CUX1 expression samples (n=156; P=.03, P=.04).Altogether, our results show that reduced CUX1 expression in myeloid cancer cells is associated both with a decrease in DNA repair efficiency and an increase in somatic mutations. We propose that genetic and epigenetic changes that reduce CUX1 expression cause a DNA repair defect that leads to the accumulation of somatic mutations and ultimately, shorter patient survival. DisclosuresSekeres:Celgene: Membership on an entity's Board of Directors or advisory committees.

Silencing the COPB2 gene decreases the proliferation, migration and invasion of human triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is highly invasive, has a high rate of recurrence and is associated with a poor clinical outcome when compared with non-TNBC due to a lack of effective and targeted treatments. The coatomer protein complex subunit β2 (COPB2) is upregulated in various types of malignant cancer. The present study demonstrated that COPB2 expression levels were significantly upregulated in breast carcinoma HS-578T cells (clonal cells originating from TNBC) when compared with non-TNBC MCF-7 cells. HS-578T cells also exhibited higher rates of proliferation, invasion and transendothelial migration when compared with MCF-7 cells. Moreover, it was identified that genetically silencing the COPB2 gene using a lentivirus-short hairpin RNA inhibited the proliferative, colony formation, migratory and invasive properties of the TNBC HS-578T cells. Mediation of the COPB2 silencing effect may be associated with regulating the phosphorylation of serine/threonine kinase AKT in the PI3K/AKT signaling pathway. These results suggested the importance of COPB2 in promoting the proliferation of TNBC cells and identified COPB2 as a potential novel therapeutic target.