SUM149 Cells Research Articles

NDR1 increases NOTCH1 signaling activity by impairing Fbw7 mediated NICD degradation to enhance breast cancer stem cell properties

BackgroundThe existence of breast cancer stem cells (BCSCs) causes tumor relapses, metastasis and resistance to conventional therapy in breast cancer. NDR1 kinase, a component of the Hippo pathway, plays important roles in multiple biological processes. However, its role in cancer stem cells has not been explored. The purpose of this study was to investigate the roles of NDR1 in modulating BCSCs.MethodsThe apoptosis was detected by Annexin V/Propidium Iodide staining and analyzed by flow cytometry. BCSCs were detected by CD24/44 or ALDEFLUOR staining and analyzed by flow cytometry. The proliferation ability of BCSCs was evaluated by sphere formation assay. The expression of interested proteins was detected by western blot analysis. The expression of HES-1 and c-MYC was detected by real-time PCR. Notch1 signaling activation was detected by luciferase reporter assay. Protein interaction was evaluated by immunoprecipitation. Protein degradation was evaluated by ubiquitination analysis. The clinical relevance of NDR1 was analyzed by Kaplan–Meier Plotter.ResultsNDR1 regulates apoptosis and drug resistance in breast cancer cells. The upregulation of NDR1 increases CD24low/CD44high or ALDEFLUORhigh population and sphere-forming ability in SUM149 and MCF-7 cells, while downregulation of NDR1 induces opposite effects. NDR1 increased the expression of the Notch1 intracellular domain (NICD) and activated the transcription of its downstream target (HES-1 and c-MYC). Critically, both suppression of Notch pathway activation by DAPT treatment or downregulation of Notch1 expression by shRNA reverses NDR1 enhanced BCSC properties. Mechanically, NDR1 interactes with both NICD or Fbw7 in a kinase activity-independent manner. NDR1 reduces the proteolytic turnover of NICD by competing with Fbw7 for NICD binding, thereby leading to Notch pathway activation. Furthermore, NDR1 might function as a hub to modulate IL-6, TNF-α or Wnt3a induced activation of Notch1 signaling pathway and enrichment of breast cancer stem cells. Moreover, we find that the elevation of NDR1 expression predictes poor survival (OS, RFS, DMFS and PPS) in breast cancer.ConclusionOur study revealed a novel function of NDR1 in regulating BCSC properties by activating the Notch pathway. These data might provide a potential strategy for eradicating BCSC to overcome tumor relapses, metastasis and drug resistance.

Targeting Estrogen Non‐Genomic Signaling in a Three‐Dimensional (3D) in VitroModel of Inflammatory Breast Cancer

Inflammatory Breast Cancer (IBC) is the most aggressive form of breast cancer with a low overall 5‐year survival rate of ~30‐60%. To this date the molecular signature associated with IBC has not been fully characterized, which lead to a lack of effective targeted therapeutics, especially for those patients that account for the 20‐40% of triple‐negative (TNBC) IBC cases. Several studies established that molecular signature of IBC shows important differences from the other breast cancer subtypes, such as overexpression of ErbB receptors, RhoC GTPase and Cox‐2, E‐cadherin aberrant expression, and loss of WISP3, among others. However, these molecular alterations do not explain completely the aggressive and rapid IBC progression. Recently, several studies have shown that estrogen can exert non‐genomic effect in triple‐negative IBC and other TNBCs, mediated by the expression of alternate estrogen receptors, including ERα36 and GPR30. Estrogen non‐genomic signaling refers to the rapid action of these alternate estrogen receptors that upon estrogen binding activates protein‐kinase cascades in the cytoplasm. We hypothesize that estrogen can elicit a specific non‐genomic signaling cascade that promotes the acquisition of aggressive oncogenic phenotypes in IBC and that targeting this pathway can be an effective anticancer therapy for IBC. To test this hypothesis, we used pharmacological inhibitors of the Epidermal Growth Factor Receptor (EGFR) alone and GPR30 alone or in combination. The first approach was to determine if inhibiting estrogen non‐genomic signaling by targeting one of the alternate receptors, GPR30, affects cell proliferation in a 3D colony formation assay using Matrigel. Preliminary observations showed that after G15 treatment (GPR30 inhibitor), there was a slight reduction in cell proliferation compared to E2, but when combined with IRESSA (EGFR inhibitor) there was a further decreased on proliferation when compared to IRESSA alone. Ongoing experiments include the effects of Icaritin (modulator of ERα36) and knockdown of ERα36 using siRNA experiments to test the effects in cell proliferation, migration and invasion. Also, we are in the process of determining the half‐maximal inhibitory concentration (IC50) of G15, IRESSA and IRESSA + G15 by performing a dose response curve (Alamar Blue cell viability assay) of SUM149 cell line, to observe if there is an additive effect when combined the pharmacological inhibitors of EGFR and GPR30. This study will provide the foundation for further characterization of the role estrogen non‐genomic signaling in the progression of IBC and to test its potential as a therapeutic target.

Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression.

Simple SummarySpecific drug targets for breast ductal carcinoma in situ (DCIS) remain elusive, despite increasing disease prevalence and burden to healthcare services. Estrogen receptor (ER)-negative HER2-positive DCIS, associated with the poorest patient prognosis, is in particular need of novel therapeutic avenues. This report provides the first evidence that a cell surface protein called JAM-A is upregulated on human DCIS patient tissues and can be readily targeted by a novel JAM-A-binding peptide inhibitor in separate in vivo models of DCIS. The anti-tumor efficacy and lack of systemic toxicity of this lead inhibitor, coupled with early indications of potential signaling pathways implicated, support the value of future studies investigating JAM-A as a novel drug target in DCIS patients.Breast ductal carcinoma in situ (DCIS) is clinically challenging, featuring high diagnosis rates and few targeted therapies. Expression/signaling from junctional adhesion molecule-A (JAM-A) has been linked to poor prognosis in invasive breast cancers, but its role in DCIS is unknown. Since progression from DCIS to invasive cancer has been linked with overexpression of the human epidermal growth factor receptor-2 (HER2), and JAM-A regulates HER2 expression, we evaluated JAM-A as a therapeutic target in DCIS. JAM-A expression was immunohistochemically assessed in patient DCIS tissues. A novel JAM-A antagonist (JBS2) was designed and tested alone/in combination with the HER2 kinase inhibitor lapatinib, using SUM-225 cells in vitro and in vivo as validated DCIS models. Murine tumors were proteomically analyzed. JAM-A expression was moderate/high in 96% of DCIS patient tissues, versus 23% of normal adjacent tissues. JBS2 bound to recombinant JAM-A, inhibiting cell viability in SUM-225 cells and a primary DCIS culture in vitro and in a chick embryo xenograft model. JBS2 reduced tumor progression in in vivo models of SUM-225 cells engrafted into mammary fat pads or directly injected into the mammary ducts of NOD-SCID mice. Preliminary proteomic analysis revealed alterations in angiogenic and apoptotic pathways. High JAM-A expression in aggressive DCIS lesions and their sensitivity to treatment by a novel JAM-A antagonist support the viability of testing JAM-A as a novel therapeutic target in DCIS.

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 P4-02-04: Endocrine therapy treatment radiosensitizes estrogen receptor-positive breast cancers

Abstract Purpose: Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of breast cancer (BC) pathogenesis and therefore a target of first-line therapies for ER-positive (ER+) BC patients. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of ER+ BC, the sequencing of therapy and the effect of ET on tumor radiosensitization remain unclear. Recently, this question has become much more clinically relevant when many physicians started offering ET as a bridging strategy to surgery and RT during the COVID-19 pandemic. Here we assessed the efficacy and mechanism of ER inhibition in ER+ BC in combination with RT in preclinical models. Methods: Clonogenic survival assays were used to assess radiosensitization. Inhibition of ER signaling was accomplished by treating ER+ MCF-7 and T47D cells with the selective ER modulator (SERM), tamoxifen, or the selective ER degrader (SERD), fulvestrant. The ER-negative SUM-159 cells were used as a negative control. DNA damage was assessed by the neutral comet assay. Efficiency of homologous recombination (HR) was measured by Rad51 foci or a GFP reporter system. Non-homologous end joining (NHEJ) efficiency was assessed with a pEYFP reporter. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow cytometry. Senescence was measured using β-galactosidase staining. Western blotting was used to quantify expression of proteins and phospho-proteins involved in cell cycle and apoptosis. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo. Synergy was determined using the fractional tumor volume (FTV) method. Results: ER inhibition with tamoxifen radiosensitized ER+ MCF-7 (10-250 nM, enhR: 1.14-1.50) and T47D (500 nM-2.0 µM, enhR: 1.33-1.60) cells but not ER-negative SUM-159 cells (500 nM-2.0 µM, enhR: 0.99-1.02). ER degradation with fulvestrant had similar radiosensitization effects in MCF-7 (1-25 nM, enhR: 1.33-1.76) and T47D cells (0.5-5 nM, enhR: 0.97-2.81) with no radiosensitization observed in SUM-159 cells (1-25 nM, enhR: 1.01-1.03). MCF-7 cells treated with 500 nM tamoxifen and 4 Gy RT had an increase in dsDNA breaks compared to RT alone as measured by the comet assay (p<0.05), and there was a decrease in NHEJ-mediated repair with tamoxifen treatment (p<0.05). No changes were observed in HR-mediated repair by Rad51 foci or an HR reporter (p=NS). RT alone and in combination with tamoxifen and fulvestrant induced similar levels of cell cycle arrest, suggesting that radiosensitization with the combination therapy is a cell-cycle independent effect. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with endocrine therapy, RT, or the combination (p=NS). Although treatment with ET did induce senescence in ER+ MCF-7 and T47D cells, the combination treatment of ET with RT induced senescence to a much greater level suggesting this mechanism may contribute to radiosensitization (p<0.05). In vivo, combination RT and tamoxifen led to a significant delay in time to tumor doubling (17 days in control, 40 days with tamoxifen alone, 32 days with RT alone, and undefined with combination; p<0.0001) and a significant difference in tumor growth between mice treated with tamoxifen or RT alone compared to mice treated with tamoxifen and RT with synergy noted with combination treatment (FTV 1.297). Conclusion: Our data suggest that ET can radiosensitize ER+ breast tumors, and ET with RT may be more effective for radiosensitization. Ongoing studies will address concurrent versus sequential ET with RT. This work also supports further clinical investigation of the timing of RT for patients receiving ET, especially as ET prior to RT is increasingly used as a bridging therapy during the COVID-19 pandemic. Citation Format: Anna R Michmerhuizen, Lynn Lerner, Andrea M Pesch, Connor Ward, Rachel Schwartz, Kari Wilder-Romans, Meilan Liu, Bryan Wharram, Alexis Harold, Ruth Azaria, Nicole Zambrana Garcia, Daniel F Hayes, James M Rae, Lori J Pierce, Corey W Speers. Endocrine therapy treatment radiosensitizes estrogen receptor-positive breast cancers [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 P4-02-04.

Abstract P5-11-02: Humoral immune responses direct breast cancer towards a basal-like phenotype and increases stemness and invasive potential

Abstract Introduction: A large subset of triple negative breast cancers (TNBC) are characterized by a high degree of lymphocytic infiltration. Previous studies have shown that TNBC contain higher densities of B lymphocytes than other breast cancer subtypes. B lymphocytes infiltration occurs early in breast cancer and is associated with microinvasion. Although the exact role of B cell in growth of TNBC is unclear, B cells have been shown to induce chronic inflammatory responses in other types of cancer such as melanoma through the activation of NF-KB. Chronic inflammation is known to increase stem cell features and promote epithelial mesenchymal transition (EMT) in cancer. Therefore, we sought to examine interactions between B cells and TNBC and determine the effects on stemness and phenotype of breast cancer cells. Methods: CD20 expression was evaluated in tissue microarrays of ER- DCIS by immunohistochemistry. To mimic the B cell rich tumor microenvironment, co-culture studies were performed with the TNBC cell line SUM159 and EBV-transformed B cells using FalconTM Cell Culture Insert with a 0.4 um filter. Expression of stemness genes in tumor cells after co-culture was evaluated using Human pluripotent stem cell kit array (Qiagen). Expression of EMT and stemness genes and markers were determined by qPCR and western blotting. Self-renewal was assessed using mammosphere assays of co-cultured cells compared to controls. Invasion assays were performed in SUM159 and MCF-12A cell lines after co-culture using Corning® BiocoatTM cell culture inserts. Results: High densities of B cells were found in 9 of 36 DCIS (25%) and associated with microinvasion (p=.035). A 2-fold increase in invasion was observed in co-cultured cells compared to controls. RTqPCR showed increased expression of stemness genes OCT4 and Nanog (1.3- and 2.3-fold change respectively). Additionally, increased protein expression of stemness genes (Snail, SOX17, α-Fetoprotein AFP, Otx2, Nanog, Sox2, Oct4 and pdx-1) was observed in co-cultured cells compared to the control in the commercial array analysis. Likewise, we observed a 2- fold increase in ALDH1A3 gene expression which correlated with an increase in ALDH1A3 protein expression in co-cultured cells compared to controls. Additionally, co-cultured cells exhibited a two-fold increase in mammosphere formation compared to controls indicating an increase in self-renewal. A 7-fold increase in gene expression in the mesenchymal marker vimentin and a slight increase expression of snail was observed. This change correlated with a decrease in gene and protein expression of E-cadherin indicating an increase in EMT in co-cultured cells. Conclusion: Our study confirms the finding that B cell infiltration in stage 0 breast cancer is associated to microinvasion. Further, our in vitro studies indicate that interactions between B cells and TNBC may lead to increased invasion, stemness, and EMT. Thus, early B cell infiltration may be a driver of aggressive disease. Further study is needed to examine cross-talk between B cells and TNBC which may identify new targets for therapeutic intervention. Additionally, studies should examine CD20+ B cell infiltration as a prognostic marker for ER- DCIS progression. Citation Format: Jennifer Sims Mourtada, Steficah Maosa, Lynn Opdenaker, Nicole Toney. Humoral immune responses direct breast cancer towards a basal-like phenotype and increases stemness and invasive potential [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 P5-11-02.

The Lipid Droplet Knowledge Portal: A resource for systematic analyses of lipid droplet biology.

Lipid droplets (LDs) are organelles of cellular lipid storage with fundamental roles in energy metabolism and cell membrane homeostasis. There has been an explosion of research into the biology of LDs, in part due to their relevance in diseases of lipid storage, such as atherosclerosis, obesity, type 2 diabetes, and hepatic steatosis. Consequently, there is an increasing need for a resource that combines datasets from systematic analyses of LD biology. Here, we integrate high-confidence, systematically generated human, mouse, and fly data from studies on LDs in the framework of an online platform named the "Lipid Droplet Knowledge Portal" (https://lipiddroplet.org/). This scalable and interactive portal includes comprehensive datasets, across a variety of cell types, for LD biology, including transcriptional profiles of induced lipid storage, organellar proteomics, genome-wide screen phenotypes, and ties to human genetics. This resource is a powerful platform that can be utilized to identify determinants of lipid storage.

Quercus acutissima Carruth. root extract triggers apoptosis, autophagy and inhibits cell viability in breast cancer cells

Ethnopharmacological relevanceThe bark of Quercus acutissima Carruth. (QA) has long been used by Chinese people to treat noncancerous growths and cancerous ailments. It was traditionally used by Chinese folk to inhibit tumor proliferation in cancerous treatment, but the specific mechanism remain to be elucidated. Aim of the studyThis study investigated the anticancer activities of QA root extract and its regulatory pathways in two human breast cancer cell lines (MCF-7 and SUM159). Materials and methodsDried QA root barks were extracted by ethanol and used to treat human breast cancer MCF-7 and SUM159 cells with varying concentrations. The CCK-8 assay, Hoechst 33342 staining assay and wound healing assay were used to detect the cell proliferation, apoptotic cell morphology, and cell migration in each group, respectively. Caspase 3 activity assay kit was used to determine caspase 3 activity. Western blot was used to measure proteins expression level in apoptosis and autophagy pathways (Bcl-W, caspase 3, Beclin1, LC3 and Atg5). LC-MS was performed to determine the chemical components in QA root extract. ResultsCCK-8 assay showed that QA root extract significantly inhibited cell viability and proliferation in breast cancer cells by a concentration-dependent manner. Cell wound healing assay indicated that it had high suppression ability on cell migration both in MCF-7 and SUM159 cells. QA root extract treatment induced the morphological and nuclear structural changes in breast cancer cells including rounded appearance and shrunken nucleus with several nuclear body fragments. Western blot indicated that QA root extract induced mitochondria-mediated apoptosis by up-regulating caspase 3 and down-regulating Bcl-W. Moreover, QA root extract up-regulated Beclin1 and Atg5, and activated LC3 in two human breast cancer cell lines. LC-MS results showed that QA root extract contains high content of bioactive compounds like coumarins and derivatives, prenol lipids, flavonoids and tannins. ConclusionsQA root extract inhibited cell proliferation and migration in MCF-7 and SUM159 cells, and it also induced cell morphology changes and regulated mitochondria-mediated apoptotic cell death and autophagic cell death.

Unlabeled aspirin as an activatable theranostic MRI agent for breast cancer.

Rationale: Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is emerging as an alternative to gadolinium-based contrast MRI. We have evaluated the possibility of CEST MRI of orthotopic breast tumor xenografts with unlabeled aspirin's conversion to salicylic acid (SA) through various enzymatic activities, most notably inhibition of cyclooxygenase (COX)-1/-2 enzymes.Methods: We measured the COX-1/-2 expression in four breast cancer cell lines by Western Blot analysis and selected the highest and lowest expressing cell lines. We then performed CEST MRI following aspirin treatment to detect SA levels and ELISA to measure levels of downstream prostaglandin E2 (PGE2). We also injected aspirin into the tail vein of mice growing orthotopic tumor xenografts which expressed high and low COX-1/-2 and acquired SA CEST MR images of these tumor xenografts for up to 70 minutes. Tumors were then harvested to perform Western Blot and ELISA experiments to measure COX-1/-2 expression and PGE2 levels, respectively.Results: Western Blots determined that SUM159 cells contained significantly higher COX-1/-2 expression levels than MDA-MB-231 cells, in line with higher levels of downstream PGE2. SA CEST MRI yielded similar contrast at approximately 3% for both cell lines, independent of COX-1/-2 expression level. PGE2 levels decreased by about 50% following aspirin treatment. Results from our mouse study aligned with cultured cells, the overall SA CEST MRI contrast in both MDA-MB-231 and SUM159 tumor xenograft models was 5~8% at one hour post injection. PGE2 levels were ten times higher in SUM159 than MDA-MB-231 and decreased by 50%. The CEST contrast directly depended on the injected dose, with ~6%, ~3% and ~1.5% contrast observed following injection of 100 µL of 300 mM, 200 mM and 150 mM aspirin, respectively.Conclusions: Our data demonstrate the feasibility of using aspirin as a noninvasive activatable CEST MRI contrast agent for breast tumor detection.

Elevated NRAS expression during DCIS is a potential driver for progression to basal-like properties and local invasiveness

BackgroundDuctal carcinoma in situ (DCIS) is the most common type of in situ premalignant breast cancers. What drives DCIS to invasive breast cancer is unclear. Basal-like invasive breast cancers are aggressive. We have previously shown that NRAS is highly expressed selectively in basal-like subtypes of invasive breast cancers and can promote their growth and progression. In this study, we investigated whether NRAS expression at the DCIS stage can control transition from luminal DCIS to basal-like invasive breast cancers.MethodsWilcoxon rank-sum test was performed to assess expression of NRAS in DCIS compared to invasive breast tumors in patients. NRAS mRNA levels were also determined by fluorescence in situ hybridization in patient tumor microarrays (TMAs) with concurrent normal, DCIS, and invasive breast cancer, and association of NRAS mRNA levels with DCIS and invasive breast cancer was assessed by paired Wilcoxon signed-rank test. Pearson’s correlation was calculated between NRAS mRNA levels and basal biomarkers in the TMAs, as well as in patient datasets. RNA-seq data were generated in cell lines, and unsupervised hierarchical clustering was performed after combining with RNA-seq data from a previously published patient cohort.ResultsInvasive breast cancers showed higher NRAS mRNA levels compared to DCIS samples. These NRAShigh lesions were also enriched with basal-like features, such as basal gene expression signatures, lower ER, and higher p53 protein and Ki67 levels. We have shown previously that NRAS drives aggressive features in DCIS-like and basal-like SUM102PT cells. Here, we found that NRAS-silencing induced a shift to a luminal gene expression pattern. Conversely, NRAS overexpression in the luminal DCIS SUM225 cells induced a basal-like gene expression pattern, as well as an epithelial-to-mesenchymal transition signature. Furthermore, these cells formed disorganized mammospheres containing cell masses with an apparent reduction in adhesion.ConclusionsThese data suggest that elevated NRAS levels in DCIS are not only a marker but can also control the emergence of basal-like features leading to more aggressive tumor activity, thus supporting the therapeutic hypothesis that targeting NRAS and/or downstream pathways may block disease progression for a subset of DCIS patients with high NRAS.

The FoxQ1 transcription factor is a novel regulator of electron transport chain complex I subunits in human breast cancer cells.

The FoxQ1 is an oncogenic transcription factor that is overexpressed in basal-like and luminal-type human breast cancers when compared to the normal mammary tissue. The FoxQ1 is implicated in mammary tumor progression. However, the mechanism by which FoxQ1 promotes mammary tumorigenesis is not fully understood. In this study, we present experimental evidence for a novel function of FoxQ1 in the regulation of complex I activity of the electron transport chain. The RNA-seq data from FoxQ1 overexpressing basal-like SUM159 cells revealed a statistically significant increase in the expression of complex I subunits NDUFS1 and NDUFS2 when compared to the empty vector (EV) transfected control cells. Consistent with these results, the basal and ATP-linked oxygen consumption rates were significantly increased by FoxQ1 overexpression in SUM159 and luminal-type MCF-7 cells. The FoxQ1 overexpression in both cell lines resulted in increased intracellular levels of pyruvate, lactate, and ATP that was associated with overexpression of pyruvate dehydrogenase and pyruvate carboxylase proteins. Activity and assembly of complex I were significantly enhanced by FoxQ1 overexpression in SUM159 and MCF-7 cells that correlated with increased mRNA and/or protein levels of complex I subunits NDUFS1, NDUFS2, NDUFV1, and NDUFV2. The chromatin immunoprecipitation assay revealed the recruitment of FoxQ1 at the promoters of both NDUFS1 and NDUFV1. The cell proliferation of SUM159 and MCF-7 cells was increased significantly by overexpression of NDUFS1 as well as NDUFV1 proteins. In conclusion, we propose that increased complex I-linked oxidative phosphorylation is partly responsible for oncogenic role of FoxQ1 at least in human breast cancer cells.

Omeprazole suppresses aggressive cancer growth and metastasis in mice through promoting Snail degradation.

Omeprazole is a proton pump inhibitor that has recently been reported to exhibit anticancer activity against several types of cancer. However, the anticancer mechanisms of omeprazole remain elusive. Snail is an oncogenic zinc finger transcription factor; aberrant activation of Snail is associated with the occurrence and progression of cancer. In this study, we investigated whether Snail acted as a direct anticancer target of omeprazole. We showed that omeprazole displayed a high binding-affinity to recombinant Snail protein (Kd = 0.076 mM), suggesting that omeprazole directly and physically binds to the Snail protein. We further revealed that omeprazole disrupted CREB-binding protein (CBP)/p300-mediated Snail acetylation and then promoted Snail degradation through the ubiquitin-proteasome pathway in HCT116 cells. Omeprazole treatment markedly suppressed Snail-driven epithelial-mesenchymal transition (EMT) in aggressive HCT116, SUM159, and 4T1 cancer cells in vitro and reduced EMT-associated tumor invasion and metastasis in cancer cell xenograft models. Omeprazole also inhibited tumor growth by limiting Snail-dependent cell cycle progression. Overall, this study, for the first time, identifies Snail as a target of omeprazole and reveals a novel mechanism underlying the therapeutic effects of omeprazole against cancer. This study strongly suggests that omeprazole may be an excellent auxiliary drug for treating patients with malignant tumors.

Antitumor properties of novel sesquiterpene lactone analogs as NFκB inhibitors that bind to the IKKβ ubiquitin-like domain (ULD).

Melampomagnolide B (MMB, 3) is a parthenolide (PTL, 1) based sesquiterpene lactone that has been used as a template for the synthesis of a plethora of lead anticancer agents owing to its reactive C-10 primary hydroxyl group. Such compounds have been shown to inhibit the IKKβ subunit, preventing phosphorylation of the cytoplasmic IκB inhibitory complex. The present study focuses on the synthesis and invitro antitumor properties of novel benzyl and phenethyl carbamates of MMB (7a-7k). Screening of these MMB carbamates identified analogs with potent growth inhibition properties against a panel of 60 human cancer cell lines (71% of the molecules screened had GI50 values<2μM). Two analogs, the benzyl carbamate 7b and the phenethyl carbamate7k, were the most active compounds. Lead compound 7b inhibited cell proliferation in M9 ENL AML cells, and in TMD-231, OV-MD-231 and SUM149 breast cancer cell lines. Interestingly, mechanistic studies showed that 7b did not inhibit p65 phosphorylation in M9 ENL AML and OV-MD-231cells, but did inhibit phophorylation of both p65 and IκBα in SUM149cells. 7b also reduced NFκB binding to DNA in both OV-MD-231 and SUM149cells. Molecular docking studies indicated that 7b and 7k are both predicted to interact with the ubiquitin-like domain (ULD) of the IKKβ subunit. These data suggest that in SUM149cells, 7b is likely acting as an allosteric inhibitor of IKKβ, whereas in M9 ENL AML and OV-MD-231cells 7b is able to inhibit an event after IκB/p65/p50 phosphorylation by IKKβ that leads to inhibition of NFκB activation and reduction in NFκB-DNA binding. Analog 7b was by far the most potent compound in either carbamate series, and was considered an important lead compound for further optimization and development as an anticancer agent.

Inhibition of Estrogen Receptor Signaling as a Strategy for Radiosensitization of ER+ Breast Cancers

Estrogen receptor (ER) expression is present in over 80% of breast tumors and has been shown to be a significant driver of tumor initiation and progression and therefore a target of first-line therapies for ER-positive (ER+) breast cancer (BC) patients. While both ionizing radiation (RT) and endocrine therapies (ET) are used for the treatment of women with ER+ BC, the sequencing of therapy and the effect of ET on tumor radiosensitization remains unclear. Here we assessed the efficacy and mechanism of ER inhibition in ER+ BC in combination with RT in multiple preclinical models.Clonogenic survival assays were used to assess radiosensitization. ER inhibition was accomplished with tamoxifen and the selective ER degrader (SERD), fulvestrant, in ER+ MCF-7 and T47D cells or ER-negative (ER-) SUM-159 cells. DNA damage was assessed by yH2AX foci. Efficiency of homologous recombination (HR) or non-homologous end joining (NHEJ) was measured by RAD51 foci or using a pYFP reporter, respectively. Cell cycle effects were measured using flow cytometry with propidium iodide (PI) staining. Apoptosis was assessed by annexin V/PI via flow cytometry. Western blotting was used to quantify protein expression. An MCF-7 xenograft model was used to assess the efficacy of tamoxifen with RT in vivo where mice were pretreated with tamoxifen for one or six days prior to starting RT. Synergy was determined using the fractional tumor volume method.One-hour pretreatment with tamoxifen prior to RT resulted in radiosensitization of ER+ MCF-7 (enhR: 1.14-1.50) and T47D (enhR: 1.33-1.60) cells but not ER- SUM-159 cells (enhR: 0.99-1.02). MCF-7 and T47D cells treated with tamoxifen and RT did not alter the kinetics of dsDNA break repair as measured by yH2AX foci (P > 0.05) but demonstrated a decrease in NHEJ-mediated repair (P < 0.05). No changes were observed in HR-mediated repair by Rad51 foci (P > 0.05). While cell cycle arrest was induced at 24 hours after RT, no changes were observed with tamoxifen treatment in combination with RT. In addition, there were no significant changes in apoptosis in MCF-7 or T47D cells with treatment of tamoxifen, RT, or the combination (P > 0.05). In vivo, an MCF-7 xenograft study demonstrated a significant delay in time to tumor doubling (17 days in control, 40 days with tamoxifen, 32 days with RT, and undefined with combination; P < 0.0001) and a significant difference in tumor growth between mice treated with tamoxifen or RT alone compared to mice treated with tamoxifen and RT with synergy noted with combination treatment.Our data suggest that endocrine therapies may be effectively used to radiosensitize ER+ breast tumors. This work also supports further clinical investigation of the timing of RT for patients receiving ET as concurrent use may be more effective than sequential, especially as ET prior to RT is increasingly used as a bridging therapy during the COVID pandemic.

Investigation of a Mitochondrial to Nuclear Signaling Pathway Induced by Onc201

Onc201 is a novel therapeutic in clinical trials that displays widespread anti-cancer activity and also has been shown to act as a radiosensitizer. Previously, we showed that Onc201 and its derivatives activate a mitochondrial protease, ClpP, resulting in mitochondrial protein degradation, induction of ATF4/CHOP and cytostatic activity in a breast cancer cell line. However, how activation of ClpP, a mitochondrial protease, signals to the nucleus has not been established. We show here that Onc201 induces GDF15, a mitochondrial stress cytokine, and further delineate the upstream signaling pathways. The aim of these studies is to better understand mechanisms of mitochondrial to nuclear signaling to allow for more effective anti-cancer strategies to be developed.To monitor mitochondrial to nuclear signaling, Sum159, a breast cancer cell line, was treated with Onc201 or its derivatives, and monitored for ATF4 or GDF15 protein expression by Western blotting. Delineation of the signaling pathways involved was achieved using siRNA directed against various targets or by use of specific kinase inhibitors.Treatment of Sum159 cells with Onc201 resulted in the induction of GDF15 and ATF4 protein as early as ∼17 hours after treatment. Using siRNA knockdowns, we found that GDF15 induction is dependent upon ClpP, ATF4, and CHOP. However, knockdown of GCN2 or HRI, which are eiF2a kinases known to regulate ATF4, had no effect on GDF15, nor did siRNA against IRE1a. Because we found that Onc201 induced Erk, Akt and AMPK activity, we targeted these pathways with specific kinase inhibitors; however, none of these pathways were required for GDF15 induction. These results suggest that other pathways may be involved in the induction of ATF4 and GDF15 and these are currently under investigation.We and others have shown that Onc201 and related molecules, activate ClpP, a mitochondrial protease resulting in rapid mitochondrial protein degradation, loss of mitochondrial function, and cytostasis or apoptosis in a variety of cancer cell lines. Importantly, Onc201 shows very low toxicity to normal cell lines and is well tolerated in patients. However, additional stratagems need to be explored to improve the efficacy of Onc201. To this end, we sought to understand how mitochondrial to nuclear signaling occurs as a way to target this pathway. These results establish that ATF4/CHOP signaling is induced and this subsequently signals to induce GDF15. However, it still remains unclear how ATF4/CHOP expression is induced and this area is currently being investigated.

The mechanism of cell death induced by silver nanoparticles is distinct from silver cations

BackgroundPrecisely how silver nanoparticles (AgNPs) kill mammalian cells still is not fully understood. It is not clear if AgNP-induced damage differs from silver cation (Ag+), nor is it known how AgNP damage is transmitted from cell membranes, including endosomes, to other organelles. Cells can differ in relative sensitivity to AgNPs or Ag+, which adds another layer of complexity to identifying specific mechanisms of action. Therefore, we determined if there were specific effects of AgNPs that differed from Ag+ in cells with high or low sensitivity to either toxicant.MethodsCells were exposed to intact AgNPs, Ag+, or defined mixtures of AgNPs with Ag+, and viability was assessed. The level of dissolved Ag+ in AgNP suspensions was determined using inductively coupled plasma mass spectrometry. Changes in reactive oxygen species following AgNP or Ag+ exposure were quantified, and treatment with catalase, an enzyme that catalyzes the decomposition of H2O2 to water and oxygen, was used to determine selectively the contribution of H2O2 to AgNP and Ag+ induced cell death. Lipid peroxides, formation of 4-hydroxynonenol protein adducts, protein thiol oxidation, protein aggregation, and activation of the integrated stress response after AgNP or Ag+ exposure were quantified. Lastly, cell membrane integrity and indications of apoptosis or necrosis in AgNP and Ag+ treated cells were examined by flow cytometry.ResultsWe identified AgNPs with negligible Ag+ contamination. We found that SUM159 cells, which are a triple-negative breast cancer cell line, were more sensitive to AgNP exposure less sensitive to Ag+ compared to iMECs, an immortalized, breast epithelial cell line. This indicates that high sensitivity to AgNPs was not predictive of similar sensitivity to Ag+. Exposure to AgNPs increased protein thiol oxidation, misfolded proteins, and activation of the integrated stress response in AgNP sensitive SUM159 cells but not in iMEC cells. In contrast, Ag+ cause similar damage in Ag+ sensitive iMEC cells but not in SUM159 cells. Both Ag+ and AgNP exposure increased H2O2 levels; however, treatment with catalase rescued cells from Ag+ cytotoxicity but not from AgNPs. Instead, our data support a mechanism by which damage from AgNP exposure propagates through cells by generation of lipid peroxides, subsequent lipid peroxide mediated oxidation of proteins, and via generation of 4-hydroxynonenal (4-HNE) protein adducts.ConclusionsThere are distinct differences in the responses of cells to AgNPs and Ag+. Specifically, AgNPs drive cell death through lipid peroxidation leading to proteotoxicity and necrotic cell death, whereas Ag+ increases H2O2, which drives oxidative stress and apoptotic cell death. This work identifies a previously unknown mechanism by which AgNPs kill mammalian cells that is not dependent upon the contribution of Ag+ released in extracellular media. Understanding precisely which factors drive the toxicity of AgNPs is essential for biomedical applications such as cancer therapy, and of importance to identifying consequences of unintended exposures.

Tumor Growth Progression in Ectopic and Orthotopic Xenografts from Inflammatory Breast Cancer Cell Lines.

Xenografts can grow in immunosuppressed hosts, such as SCID mice, and tumor material can be injected into hosts either ectopically or orthotopically. Choosing the correct model to use is a crucial step in animal research. The aim of this study was to report the differences between ectopic and orthotopic xenografts in tumor progression, metastasis capacity, histological features, and steroid hormone profiles in xenografts from the cIMC (canine inflammatory mammary cancer) cell line IPC-366 and hIBC (human inflammatory breast cancer) cell line SUM149. To achieve this purpose, 40 female mice 6–8 weeks old were inoculated with IPC-366 and SUM149 cells subcutaneously (ectopic models) or into mammary fat pad (orthotopic models). Mice were monitored for tumor progression and appearance of metastases, and generated tumors were analyzed in terms of histological examination and steroid hormone production. The results revealed differences in tumor appearance and percentage of metastasis between ectopic and orthotopic models, which were higher in the ectopic xenografts from both cell lines. However, both models had similar characteristics of tumor progression, histological features, and steroid hormone secretion profiles. We show that the ectopic model can be validated as a good and useful model of tumor development in addition to, not contrary to, the orthotopic model in breast cancer research.

Targeting Lipocalin-2 in Inflammatory Breast Cancer Cells with Small Interference RNA and Small Molecule Inhibitors.

Inflammatory Breast Cancer (IBC) is an aggressive form of invasive breast cancer, highly metastatic, representing 2–4% of all breast cancer cases in the United States. Despite its rare nature, IBC is responsible for 7–10% of all breast cancer deaths, with a 5-year survival rate of 40%. Thus, targeted and effective therapies against IBC are needed. Here, we proposed Lipocalin-2 (LCN2)—a secreted glycoprotein aberrantly abundant in different cancers—as a plausible target for IBC. In immunoblotting, we observed higher LCN2 protein levels in IBC cells than non-IBC cells, where the LCN2 levels were almost undetectable. We assessed the biological effects of targeting LCN2 in IBC cells with small interference RNAs (siRNAs) and small molecule inhibitors. siRNA-mediated LCN2 silencing in IBC cells significantly reduced cell proliferation, viability, migration, and invasion. Furthermore, LCN2 silencing promoted apoptosis and arrested the cell cycle progression in the G0/G1 to S phase transition. We used in silico analysis with a library of 25,000 compounds to identify potential LCN2 inhibitors, and four out of sixteen selected compounds significantly decreased cell proliferation, cell viability, and the AKT phosphorylation levels in SUM149 cells. Moreover, ectopically expressing LCN2 MCF7 cells, treated with two potential LCN2 inhibitors (ZINC00784494 and ZINC00640089) showed a significant decrease in cell proliferation. Our findings suggest LCN2 as a promising target for IBC treatment using siRNA and small molecule inhibitors.

Caveolin-1 is a prognostic marker and suppresses the proliferation of breast cancer.

BackgroundTo explore the role of caveolin-1 (Cav-1) in breast cancer (BC).MethodsCav-1 expression data were downloaded from the Tumor Immune Estimation Resource (TIMER) and Gene Expression Omnibus (GEO) databases. We compared the expression of Cav-1 in different tumor tissues and between BC tissues and normal tissues (NTs), as well as the differences between different clinical traits. Kaplan-Meier survival analysis and univariate and multivariate Cox regression analyses were used to determine whether Cav-1 serves as a prognostic factor. The correlations of Cav-1 expression with the immune microenvironment and infiltrating immune cells were also analyzed. Quantitative polymerase chain reaction (qPCR) was used to detect Cav-1 mRNA expression in the MCF-7, SKB-R3, MDB-MB-231, and SUM-159 cell lines. LV-Cav-1-RNAi was transfected into MCF-7 and MDB-MB-231 cells, and the MTT assay was used to detect cell proliferation. Subsequently, MDB-MB-231 cells carrying the Cav-1-RNAi gene were used to determine the effects of Cav-1 knockdown on tumor growth in vivo using a severe combined immunodeficiency (SCID) model.ResultsCav-1 was enriched in most solid tumors, and its expression was lower in BC tissues than in NT. Cav-1 expression was shown to be related to patients’ clinical outcomes. Cav-1 was expressed in the MCF-7, SKB-R3, MDB-MB-231, and SUM-159 cell lines. The MTT assay revealed that the proliferative ability of MDB-MB-231 and MCF-7 cells was accelerated. The tumor volume of SCID mice administered with LV-Cav-1-RNAi cells was increased.ConclusionsThese results suggest that Cav-1 may serve as a suppressor in the development of BC.

Anti-Angiogenic Treatments Interact with Steroid Secretion in Inflammatory Breast Cancer Triple Negative Cell Lines

Simple SummaryInflammatory breast cancer (IBC) is the most aggressive breast cancer and is associated with poor prognosis. Exacerbated angiogenesis, lymphangiogenesis and lymphangiotropism are hallmarks of this tumour. Current antiangiogenic therapies have minimal effects on overall survival in IBC patients. Furthermore, it is well established that steroid hormones are strongly related to tumour development and progression, angiogenesis regulation and metastasis. We investigated the effect of different antiangiogenic therapies on steroid and angiogenic growth factor production using two inflammatory breast cancer cell lines. We reported that sex steroid hormones could regulate the production of angiogenic factors, since after the results, P4 and E2 were involved in VEGF production and androgens in the formation of vascular-like structures. Moreover, we reported that elevated intratumoural concentrations of T and E1SO4 could be associated with decreased metastatic rates and the promotion of tumour progression, respectively, and thus the measurement of sex steroids and growth factors may be useful to develop preventive and individualised therapeutic strategies.Human inflammatory breast cancer (IBC) is a highly angiogenic disease for which antiangiogenic therapy has demonstrated only a modest response, and the reason for this remains unknown. Thus, the purpose of this study was to determine the influence of different antiangiogenic therapies on in vitro and in vivo steroid hormone and angiogenic growth factor production using canine and human inflammatory breast carcinoma cell lines as well as the possible involvement of sex steroid hormones in angiogenesis. IPC-366 and SUM149 cell lines and xenotransplanted mice were treated with different concentrations of VEGF, SU5416, bevacizumab and celecoxib. Steroid hormone (progesterone, dehydroepiandrostenedione, androstenedione, testosterone, dihydrotestosterone, estrone sulphate and 17β-oestradiol), angiogenic growth factors (VEGF-A, VEGF-C and VEGF-D) and IL-8 determinations in culture media, tumour homogenate and serum samples were assayed by EIA. In vitro, progesterone- and 17β-oestradiol-induced VEGF production promoting cell proliferation and androgens are involved in the formation of vascular-like structures. In vivo, intratumoural testosterone concentrations were augmented and possibly associated with decreased metastatic rates, whereas elevated E1SO4 concentrations could promote tumour progression after antiangiogenic therapies. In conclusion, sex steroid hormones could regulate the production of angiogenic factors. The intratumoural measurement of sex steroids and growth factors may be useful to develop preventive and individualized therapeutic strategies.