Levistolide A induces endoplasmic reticulum stress-triggered apoptosis via ATF6 in triple-negative breast cancer.

To explore the molecular mechanisms of tumor-associated calcium signal transduction factor 2 (TROP2) affecting the occurrence and development of triple-negative breast cancer (TNBC). The TCGA database, immunohistochemical staining, and qRT-PCR were used to analyze the expression of TROP2 in TNBC tissues and cells. The protein expressions of TROP2 and inositol 1,4,5-trisphosphate receptor (IP3R) after TROP2 knockdown were detected by western blot (WB). Cell proliferation was detected by CCK8 and colony formation assay, Annexin V-APC/PI flow cytometry was used to detect apoptosis, and intracellular calcium ion (Ca2+) was detected by flow cytometry with Fura 2-AM fluorescent probe. Finally, the morphological changes of the endoplasmic reticulum (ER) were observed by transmission electron microscopy, and the expression of ER stress (ERS)-related proteins was detected by WB and immunofluorescence staining. TROP2 was up-regulated in TNBC tumor tissues and cells. Silencing TROP2 decreased the proliferation rate and clone formation number, and increased the apoptosis rate and the Ca2+ level in TNBC cells. These phenomena were reversed after the addition of 2-APB. In addition, after TROP2 knockdown, the expressions of IP3R and ERS-related proteins were up-regulated, the ER was cystic dilated, and ERS was activated. And the addition of 2-APB significantly inhibited the activation of ERS induced by TROP2 knockdown. TROP2 regulated the proliferation and apoptosis of TNBC cells through a Ca2+-dependent ERS signaling pathway.

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

Palmitic acid (PA) upregulates oxidized LDL receptor-1 (LOX-1), a scavenger receptor responsible for uptake of oxidized LDL (oxLDL), and enhances oxLDL uptake in macrophages. However, the precise underlying mechanism remains to be elucidated. PA is known to induce endoplasmic reticulum (ER) stress in various cell types. Therefore, we investigated whether ER stress is involved in PA-induced LOX-1 upregulation. PA induced ER stress, as determined by phosphorylation of PERK, eIF2α, and JNK, as well as induction of CHOP in macrophage-like THP-1 cells. Inhibitors [4-phenylbutyric acid (PBA), sodium tauroursodeoxycholate (TUDCA), and salubrinal] and small interfering RNA (siRNA) for the ER stress response decreased PA-induced LOX-1 upregulation. Thapsigargin, an ER stress inducer, upregulated LOX-1, which was decreased by PBA and TUDCA. We next examined whether unsaturated FAs could counteract the effect of PA. Both oleic acid (OA) and linoleic acid (LA) suppressed PA-induced LOX-1. Activation of the ER stress response observed in the PA-treated cells was markedly attenuated when the cells were cotreated with OA or LA. In addition, OA and LA suppressed thapsigargin-induced LOX-1 upregulation with reduced activation of ER stress markers. Our results indicate that activation of ER stress is involved in PA-induced LOX-1 upregulation in macrophages, and that OA and LA inhibit LOX-1 induction through suppression of ER stress.

Background: Women with triple-negative breast cancer (TNBC) have a more aggressive clinical course, with a higher propensity to metastasize and a worse outcome due to a lack of effective therapies and significant intratumoral and intertumoral heterogeneity. Development of novel therapeutic strategies represents a clear unmet need. We have developed a first-in-class compound, an oligobenzamide, ERX-41, that has anti-proliferative activity against all six molecular subtypes of TNBC. Methods: In vitro activity of ERX-41 on TNBC cell lines was tested using CellTiter glo, MTT, and apoptosis assays. Efficacy of ERX-41 was tested using TNBC patient derived explants (PDEs) ex vivo, cell line-derived xenografts (CDXs), and patient derived xenografts (PDX) in vivo. To examine the mechanism, we conducted mass spec analyses using total lysates of TNBC cells treated with vehicle or ERX-41. Results: ERX-41 demonstrated potent activity in both blocking proliferation and inducing apoptosis in 30 distinct cell line models of TNBC, (representing all six molecular subtypes of TNBC), with an IC50 that ranges from 50-250nM. Incubation of ERX-41 with PDEs from primary TNBC patient tumors ex vivo caused a significant reduction in proliferation indices, as measured by Ki67 staining. ERX-41 also decreased proliferation and increased apoptosis in explants from TNBC CDX and PDX tumors cultured ex vivo. Oral administration of ERX-41 (10 mg/kg/daily) was shown to be non-toxic and dramatically limited the growth of CDX tumors derived from MDA-MB-231, SUM-159 or D2A1 syngeneic tumors. Importantly, ERX-41 treatment also significantly reduced tumor progression in four TNBC PDX (PDX-1, PDX-89, PDX-96 and PDX-98) models compared to the vehicle treated control group. Our ultrastructural and molecular studies indicate that ERX-41 induces significant endoplasmic reticulum (ER) stress within TNBC cells but not in primary epithelial cells. Global mass spectrometry studies indicated that ERX-41 treatment resulted in the alteration [down regulation (265 proteins) or upregulation (218 proteins)] of 483 proteins out of ~4000 proteins quantified with two or more peptides. Reactome pathway analysis indicated that the top pathways modulated by ERX-41 included Intra-Golgi and retrograde Golgi-to-ER traffic, membrane trafficking and TP53 mediated apoptosis. ER stress induced by ERX-41 blocks de novo protein synthesis, and triggers ER-assisted degradation (ERAD) pathways, causing TNBC apoptotic cell death. Conclusions: ERX-41 is orally bioavailable, non-toxic, and demonstrated activity in primary PDEs, CDXs and PDXs. The ability of ERX-41 to induce ER stress and apoptotic cell death in multiple types of TNBC suggests that this drug targets a fundamental weakness in TNBC cells (the high basal level of ER stress) and can effectively overcome the heterogeneity of TNBC. These studies strongly support the further clinical translation of ERX-41. Citation Format: Suryavathi Viswanadhapalli, Xihui Liu, Shi-Hong Ma, Tae-Kyung Lee, Mengxing Li, Weiwei Tang, Junhao Liu, Xiaonan Li, Gangadhara R. Sareddy, Rajeshwar Rao Tekmal, Jung-Mo Ahn, Ratna K. Vadlamudi, Ganesh V. Raj. Preclinical evaluation of ERX-41 in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1237.

Background: Triple negative breast cancer (TNBC) is a type of aggressive breast cancer with a poor survival. Endoplasmic reticulum (ER) stress, induced by exposed to intrinsic (e.g. oncogenes) and external factors (e.g. chemotherapies), is an important factor in both tumor progression and responses to chemotherapies. Recent studies have demonstrated that the progression of ER stress may result in the emergence of cancer stem cells (CSC), which also arise as a consequence of metastasis. However, the mechanism of targeting ER stress signaling in cancer stem cells remains to be investigated. Aim: (i) To investigate the mechanistic role of Oxysterol binding protein-like 3 (OSBPL3) in TNBC and its links with breast cancer mortality, particularly caused by chemotherapeutic resistance and metastasis; and (ii) to test a hypothesis that OSBPL3 facilitates the abnormal activation of ER stress and cancer stemness, result in the resistance to chemotherapy and metastasis in TNBC. Methods: The expression level of OSBPL3 in TNBC was determined by immunohistochemical staining and the mRNA expression profiles from the TCGA database. Roles of OSBPL3 in cancer cell growth, metastasis, stemness and activation of ER stress were determined by molecular and cell biology methods. Immunoprecipitation was used to examine the binding of proteins and chromatin immunoprecipitation (ChIP), promoter luciferase reporter assay was included for detecting the transcription of OSBPL3 in TNBC. TNBC xenograft nude mice were used to study the inhibition of tumor development. Results: OSBPL3 is a novel TNBC target, which is overexpressed in TNBC tissues and significantly associated with clinical pathology of advanced TNM stage and poor prognosis of tumor patients. Mechanistically, Increased OSBPL3 induced ER stress and ER stress agonist (tunicamycin) treatment results in the translocation of XBP1, an ER stress sensor, into the nucleus to induce OSBPL3 expression through direct binding to the OSBPL3 promoter. In addition, OSBPL3 facilitates the stemness induced by ER stress via phosphorylating GSK3β and transporting accumulated β-catenin to the nucleus to promote the expression of stemness-related transcriptional factors, ultimately resulting in tumor metastasis and resistance to chemotherapy. Furthermore, OSBPL3-specific siRNAs encapsulated by jetPEI nanocarriers prominently inhibit tumor growth and epirubicin-induced resistance in vivo. Conclusion: We identified an important role of OSBPL3-ER stress-XBP1 feedback loop in the stemness and resistance to chemotherapy, representing a future biomarker and therapeutic target of TNBC. Citation Format: Shengyu Pu, Huimin Zhang, Yu Ren, Jianjun He, Na Hao. OSBPL3 facilitates endoplasmic reticulum stress and stemness in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1476.

ADAR1-Dependent RNA Editing Promotes MET and iPSC Reprogramming by Alleviating ER Stress.

A specific polymorphism in the hemochromatosis (HFE) gene, H63D, is over-represented in neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer disease. Mutations of HFE are best known as being associated with cellular iron overload, but the mechanism by which HFE H63D might increase the risk of neuron degeneration is unclear. Here, using an inducible expression cell model developed from a human neuronal cell line SH-SY5Y, we reported that the presence of the HFE H63D protein activated the unfolded protein response (UPR). This response was followed by a persistent endoplasmic reticulum (ER) stress, as the signals of UPR sensors attenuated and followed by up-regulation of caspase-3 cleavage and activity. Our in vitro findings were recapitulated in a transgenic mouse model carrying Hfe H67D, the mouse equivalent of the human H63D mutation. In this model, UPR activation was detected in the lumbar spinal cord at 6 months then declined at 12 months in association with increased caspase-3 cleavage. Moreover, upon the prolonged ER stress, the number of cells expressing HFE H63D in early apoptosis was increased moderately. Cell proliferation was decreased without increased cell death. Additionally, despite increased iron level in cells carrying HFE H63D, it appeared that ER stress was not responsive to the change of cellular iron status. Overall, our studies indicate that the HFE H63D mutant protein is associated with prolonged ER stress and chronically increased neuronal vulnerability.

Recent evidences indicate that triple-negative breast cancer (TNBC) cells with a mesenchymal phenotype show a basal activation of the unfolded protein response (UPR) that increases their sensitivity to endoplasmic reticulum (ER) stress although the underlying cell death mechanism remains largely unexplored. Here we show that both caspase-8-dependent and -independent apoptotic mechanisms are activated in TNBC cells undergoing sustained ER stress. Activation of the extrinsic apoptotic pathway by ER stress involves ATF4-dependent upregulation of tumor necrosis factor-related apoptosis-inducing ligand receptor 2 (TRAIL-R2/DR5). In addition, accumulation of BH3-only protein Noxa at the mitochondria further contributes to apoptosis following ER stress in TNBC cells. Accordingly, simultaneous abrogation of both extrinsic and intrinsic apoptotic pathways is required to inhibit ER stress-induced apoptosis in these cells. Importantly, persistent FLICE-inhibitory protein (FLIP) expression plays an adaptive role to prevent early activation of the extrinsic pathway of apoptosis upon ER stress. Overall, our data show that ER stress induces cell death through a pleiotropic mechanism in TNBC cells and suggest that targeting FLIP expression may be an effective approach to sensitize these tumor cells to ER stress-inducing agents.

From adaption to death: endoplasmic reticulum stress as a novel target of selective neurodegeneration?

H3K27ac-induced RHOXF2 activates Wnt2/β-catenin pathway by binding to HOXC13 to aggravate the malignant progression of triple negative breast cancer

Expression of mutant surfactant protein C (SFTPC) results in endoplasmic reticulum (ER) stress in type II alveolar epithelial cells (AECs). AECs have been implicated as a source of lung fibroblasts via epithelial-to-mesenchymal transition (EMT); therefore, we investigated whether ER stress contributes to EMT as a possible mechanism for fibrotic remodeling. ER stress was induced by tunicamyin administration or stable expression of mutant (L188Q) SFTPC in type II AEC lines. Both tunicamycin treatment and mutant SFTPC expression induced ER stress and the unfolded protein response. With tunicamycin or mutant SFTPC expression, phase contrast imaging revealed a change to a fibroblast-like appearance. During ER stress, expression of epithelial markers E-cadherin and Zonula occludens-1 decreased while expression of mesenchymal markers S100A4 and α-smooth muscle actin increased. Following induction of ER stress, we found activation of a number of pathways, including MAPK, Smad, β-catenin, and Src kinase. Using specific inhibitors, the combination of a Smad2/3 inhibitor (SB431542) and a Src kinase inhibitor (PP2) blocked EMT with maintenance of epithelial appearance and epithelial marker expression. Similar results were noted with siRNA targeting Smad2 and Src kinase. Together, these studies reveal that induction of ER stress leads to EMT in lung epithelial cells, suggesting possible cross-talk between Smad and Src kinase pathways. Dissecting pathways involved in ER stress-induced EMT may lead to new treatment strategies to limit fibrosis.

Successful treatment of triple negative (ER-, PR-, HER2-) breast cancers (TNBC) remains elusive because of the limitations of currently available chemotherapeutic agents for TNBC, drug resistance and drug toxicities. Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, has been shown to exhibit anticancer actions both in vitro and in vivo in a variety of cancers, including human breast cancer. Here, we report that DHA induces apoptosis in TNBC cell lines, which is associated with cleavage of caspase-8 and -9. DHA induces endoplasmic reticulum (ER) stress evidenced by the increased levels of ER stress markers, GRP78, CHOP, and spliced XBP-1, and induced increased levels of death receptor DR5 protein expression. siRNAs to CHOP and DR5 blocked DHA-induced apoptosis and siRNA to CHOP blocked DHA-induced upregulation of DR5, indicating that DHA induces ER stress mediated death receptor dependent apoptosis in TNBC cells via CHOP. Furthermore, our data show that DHA mediated increases in levels of reactive oxygen species (ROS), apoptosis and ER stress can be blocked by antioxidants; N-Acetyl Cysteine (NAC) and alpha-tocopherol (αT) indicating that ROS plays a critical role in DHA-induced apoptosis and ER stress. In contrast to αT, gamma-tocotrienol (γT3), another form of vitamin E, cooperated with DHA to induce apoptosis and ER stress in TNBC cells. siRNAs to CHOP and DR5 blocked the combination effects mediated by DHA + γT3 on induction of apoptosis and ER stress. Taken together, our data, for the first time, demonstrate that DHA induces apoptosis in TNBC cells via activation of an ER stress mediated DR5 dependent pro-apoptotic pathway. Our data also show that different forms of vitamin E exhibit different combinational effects on DHA-induced apoptosis; namely, blockage by αT and enhancement by γT3. (Funding for these studies was provided by the Clayton Foundation for Research). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4953. doi:1538-7445.AM2012-4953

Omega-3 fatty acid enriched diets especially diets rich in docosahexaenoic acid (DHA) have long been associated with a lower incidence of a variety of cancers. Both in vivo and in vitro studies have indicated that DHA exerts anticancer actions via inducing tumor cells to undergo programmed cell death or decreasing cell proliferation. In this study, we investigated the anticancer actions of DHA in human triple negative breast cancer (TNBC) cells in vitro. Data indicate that DHA induces apoptosis in MDA-MB-231 and BT-20 human p53 mutant TNBC cells by activating both intrinsic and extrinsic death-mediating pathways. Activation of apoptosis is associated with induction of endoplasmic reticulum (ER) stress, and increased levels of phosphorylated JNK (pJNK), and CHOP and DR5 protein levels; as well as, reduced levels of pIκB and NF-κB downstream mediators: c-FLIP, Survivin and Bcl-2. DHA induces ROS generation and antioxidants, RRR-alpha tocopherol (αT) and N-acetyl-Cysteine (NAC) block DHA-induced apoptosis, ER stress and upregulation of pJNK/CHOP/DR5. Taken together, our data demonstrate that DHA induces apoptosis in p53 mutant TNBC via activation of ROS mediated ER stress and suppression of NF-kB mediated anti-apoptotic factors. Since p53 mutant TNBCs are associated with poor prognosis and limited treatment options, these studies suggest DHA may be a successful adjuvant treatment strategy. Research is supported by the Clayton Foundation for Research. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2011-4765

Much attention has been directed to the study of triple-negative breast cancer (TNBC) because of its short disease-free interval from diagnosis and more aggressive course in the metastatic setting. TNBC also lacks specific target receptors for treatment. Third-generation metronomic chemotherapy using available agents that target DNA repair complexes (i.e., taxanes), and cell proliferation (i.e., anthracycline) have shown promise results. However, specific regimens for TNBC at the different disease stages have not been clearly defined. Therefore, researchers are seeking molecular biomarkers to predict response to current chemotherapeutic agents. In a previous study of TNBC, we demonstrated that low doses of anthracyclines and taxanes, which are known to increase endoplasmic reticulum stress, increased tumor cell expression of glucose-regulated protein 78 (GRP78), a key regulator of the unfolding protein response (UPR), resulting in tumor cell apoptosis. GRP78 gene expression is upregulated via CREB2L1, an endoplasmic reticulum transmembrane transcription factor and member of the CREB/ATF family of transcription factors. CREB3L1 is a metastatic suppressor and functions as a transducer of UPR. The aim of the present study was to investigate the effect of low doses of doxorubicin and paclitaxel on UPR activation in metastatic TNBC cells by determining CREB3L1 protein expression in correlation to cell-surface GRP78 expression. Furthermore, we related CREB3L1 and cell-surface GRP78 expression with the migration potential of the cells in response to treatment. We found that metronomic doses of doxorubicin significantly induced CREB3L1 and cell surface GRP78 expression in TNBC cells. CREB3L1 increased by 2.5-fold in MDAMB231 and by 3-fold in MDAMB468 (p<0.04); GRP78 increased by 3.7-fold and 6.1-fold, respectively (p<0.01). Similar results were obtained with low doses of paclitaxel. A strong correlation was observed between CREB3L1 and cell-surface GRP78 protein expression. The increased expression of both proteins in MDAMB231 (highly metastatic) and MDAMB468 (moderated metastatic) was associated with significant inhibition of the migration capacity of the treated TNBC cells. In contrast, the migration capacity of treated estrogen-positive MCF7 cells (non-metastatic) and Her2- positive BT474 cells (highly metastatic) was poorly inhibited, coinciding with a non-significant increase in CREB3L1 and GRP78 expression. The results demonstrated that the drug effect via the UPR was specific for TNBC and unrelated to the metastatic profile of the cells. This study is the first stage in the identification of UPR-related biomarkers of beneficial outcome of metronomic chemotherapy for TNBC. Future mechanistic studies should focus on the possible use of cell surface GRP78 and CREB3L1 as targets for combination therapies. Citation Format: Annat Raiter, Julia Lipovetsky, Britta Hardy, Rinat Yerushalmi. UPR biomarker expression correlates with inhibition of migration of TNBC cells treated with low doses of chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4506. doi:10.1158/1538-7445.AM2017-4506

Triple-negative breast cancer (TNBC) is the most common malignant tumor in China. The expression and cell surface levels of TNF receptor superfamily member 10B (TNFRSF10B) are associated with apoptosis and chemotherapy. However, the precise molecular mechanisms that govern the regulation of TNFRSF10B remain unclear. RNA-Seq data related to TNBC chemotherapy resistance were acquired from the GEO database. The mRNA and protein levels of TNFRSF10B were detected using RT-PCR and Western blotting, respectively. Cell Counting Kit-8 (CCK-8) and colony formation assays were used to detect cell proliferation. Annexin V/7-AAD staining was used to evaluate apoptosis. The cell membrane TNFRSF10B was analyzed by Western blotting and immunofluorescence. Inducers and inhibitors of endoplasmic reticulum stress (ERS) were used to assess the effect of ERS on TNFRSF10B localization. TNFRSF10B expression was downregulated in TNBC and was associated with prognosis. TNFRSF10B overexpression inhibits the growth of TNBC both in vivo and in vitro and can partially counteract chemotherapy resistance. ERS activation in TNBC promotes the expression of TNFRSF10B, leading to its enrichment on the cell membrane surface, thereby activating the apoptotic pathways. ERS regulates the expression and subcellular localization of TNFRSF10B in TNBC cells. They synergistically affect anti-apoptosis and chemotherapy resistance in TNBC cells.