Sensitivity Of Breast Cancer Cells Research Articles

The effect of HIF-1α inhibition in breast cancer cells prior to doxorubicin treatment under conditions of normoxia and hypoxia

BackgroundOxygen deprivation is a key hallmark within solid tumours that contributes to breast-tumour pathophysiology. Under these conditions, neoplastic cells activate several genes, regulated by the HIF-1 transcription factor, which alters the tumour microenvironment to promote survival – including resistance to cell death in therapeutic attempts such as doxorubicin (Dox) treatment. MethodsWe investigated HIF-1ɑ as a therapeutic target to sensitize breast cancer cells to Dox treatment. Under both normoxic (21% O2) and hypoxic (∼0.1% O2) conditions, the HIF-1 inhibitor, 2-methoxyestradiol (2-ME), was investigated as an adjuvant for its ability to alter MCF-7 cell viability, apoptosis, autophagy and molecular pathways which are often associated with increased cell survival. ResultsHere we observed that an inverse relationship between HIF-1ɑ and apoptosis exists and that Dox promotes autophagy under hypoxic conditions. Although adjuvant therapy with 2-ME induced an antagonistic effect in breast cancer cells, upregulated HIF-1ɑ expression in a hypoxic environment promotes treatment resistance and this was attenuated once HIF-1ɑ gene expression was silenced. ConclusionTherefore, highlighting the identification of possible hypoxia-targeting therapies for breast cancer patients can be beneficial by promoting more favourable treatment responses.

18F-FDG Micro PET/CT imaging to evaluate the effect of BRCA1 knockdown on MDA-MB231 breast cancer cell radiosensitivity

ObjectiveRadioresistance of tumor cells is a major factor associated with failure of radiotherapy (RT). This study aimed to investigate the effect of BRCA1 knockdown on MDA-MB231 breast cancer cell radiosensitivity. Materials and methodsShort hairpin RNA (shRNA) was used to knockdown BRCA1 gene in MDA-MB231 cells. Cell viability and proliferative capacity were assessed by CCK-8 and colony formation assays, respectively. We established xenograft models in nude mice to evaluate tumor volume and tumor weight. The mice were imaged by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) before and after RT to evaluate changes in maximum standardized uptake value (SUVmax) and tumor SUVmax/muscle SUVmax (TMR). Changes in HIF-1α, Glut-1 and Ki-67 were analyzed and the correlation between 18F-FDG uptake and tumor biology was analyzed. ResultsCompared with the control cells, RT significantly reduced cell viability and colony formation capacity in cells with the BRCA1 gene knockdown. In vivo assays showed that there was obvious delay in the tumor growth in the shBRCA1+RT group compared with the control group. 18F-FDG Micro PET/CT indicated a reduction in glucose metabolism in the shBRCA1+RT group, with statistically significant differences in both the SUVmax and TMR. The data showed the expression of HIF-1α, Glut-1 and Ki-67 was downregulated in the shBRCA1+RT group, and both SUVmax and TMR had significant correlation with tumor biology. ConclusionThese results demonstrated that BRCA1 knockdown improves the sensitivity of MDA-MB231 breast cancer cells to RT. In addition, 18F-FDG PET/CT imaging allows non-invasive analysis of tumor biology and assessment of radiosensitivity.

Glycosylated modification of MUC1 maybe a new target to promote drug sensitivity and efficacy for breast cancer chemotherapy

Breast cancer, the most common cancer in women, usually exhibits intrinsic insensitivity to drugs, even without drug resistance. MUC1 is a highly glycosylated transmembrane protein, overexpressed in breast cancer, contributing to tumorigenesis and worse prognosis. However, the molecular mechanism between MUC1 and drug sensitivity still remains unclear. Here, natural flavonoid apigenin was used as objective due to the antitumor activity and wide availability. MUC1 knockout (KO) markedly sensitized breast cancer cells to apigenin cytotoxicity in vitro and in vivo. Both genetical and pharmacological inhibition significantly enhanced the chemosensitivity to apigenin and clinical drugs whereas MUC1 overexpression conversely aggravated such drug resistance. Constitutively re-expressing wild type MUC1 in KO cells restored the drug resistance; however, the transmembrane domain deletant could not rescue the phenotype. Notably, further investigation discovered that membrane-dependent drug resistance relied on the extracellular glycosylated modification since removing O-glycosylation via inhibitor, enzyme digestion, or GCNT3 (MUC1 related O-glycosyltransferase) knockout markedly reinvigorated the chemosensitivity in WT cells, but had no effect on KO cells. Conversely, inserting O-glycosylated sites to MUC1-N increased the drug tolerance whereas the O-glycosylated deletant (Ser/Thr to Ala) maintained high susceptibility to drugs. Importantly, the intracellular concentration of apigenin measured by UPLC and fluorescence distribution firmly revealed the increased drug permeation in MUC1 KO and BAG-pretreated cells. Multiple clinical chemotherapeutics with small molecular were tested and obtained the similar conclusion. Our findings uncover a critical role of the extracellular O-glycosylation of MUC1-N in weakening drug sensitivity through acting as a barrier, highlighting a new perspective that targeting MUC1 O-glycosylation has great potential to promote drug sensitivity and efficacy.

Effect of long noncoding RNA CCAT2 on drug sensitivity to 5-fluorouracil of breast cancer cells through microRNA-145 meditated by p53.

The current study was set out to investigate the mechanism by which silenced long noncoding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2) modulates the cell growth, migration, invasion, and drug sensitivity of breast cancer (BC) cells to 5-fluorouracil (5-Fu) with the involvement of miR-145 and p53. First, high CCAT2 expression was presented in BC cells and tissues. Subsequently, the links between CCAT2 expression and BC clinicopathological features were analyzed. Highly-expressed CCAT2 was linked to lymph node metastasis, positive progesterone receptor, estrogen receptor, and Ki-67 of BC cells. Then, the gain- and loss-of-function approaches were performed to measure the regulatory role of CCAT2 in the biological processes of BC cells. Silencing of CCAT2 suppressed in vitro cell growth, proliferation, invasion, migration abilities, and epithelial-mesenchymal transformation, increased cell apoptosis, and enhanced drug sensitivity of BC cells. Silencing of CCAT2 upregulated miR-145, which was poorly expressed in drug-resistant BC cells. p53 can bind to the miR-145 promoter region and increase miR-145 expression. Upregulation of miR-145 induced by silencing of CCAT2 can be invalidated by p53-siRNA. To conclude, p53-induced activation of miR-145 could be inhibited by CCAT2, while overexpression of CCAT2 could improve the drug resistance of BC cells to 5-Fu.

Mitochondrial ribosomal small subunit (MRPS) MRPS23 protein-protein interaction reveals phosphorylation by CDK11-p58 affecting cell proliferation and knockdown of MRPS23 sensitizes breast cancer cells to CDK1 inhibitors.

Post-translational modification of some mitoribosomal proteins has been found to regulate their functions. MRPS23 has been reported to be overexpressed in various cancers and has been predicted to be involved in increased cell proliferation. Furthermore, MRPS23 is a driver of luminal subtype breast cancer. However, its exact role and function in cancer remains unknown. METHODS ANDRESULTS: Our previous study identified protein-protein interactions involving MRPS23 and CDK11A. In this study, we confirmed the interaction of MRPS23 with the p110 and p58 isoforms of CDK11A. Phosphoprotein enrichment studies and in vitro kinase assay using CDK11A/cyclin D3 followed by MALDI-ToF/ToF analysis confirmed the phosphorylation of MRPS23 at N-terminal serine 11 residue. Breast cancer cells expressing the MRPS23 (S11G) mutant showed increased cell proliferation, increased expression of PI3-AKT pathway proteins [p-AKT (Ser47), p-AKT (Thr308), p-PDK (Ser241) and p-GSK-3β (Ser9)] and increased antiapoptotic pathway protein expression [Bcl-2, Bcl-xL, p-Bcl2 (Ser70) and MCL-1] when compared with the MRPS23 (S11A) mutant-overexpressing cells. This finding indicated the role of MRPS23 phosphorylation in the proliferation and survival of breast cancer cells. The correlation of inconsistent MRPS23 phosphoserine 11 protein expression with CDK11A in the breast cancer cells suggested phosphorylation by other kinases. In vitro kinase assay showed that CDK1 kinase also phosphorylated MRPS23 and that inhibition using CDK1 inhibitors lowered phospho-MRPS23 (Ser11) levels. Additionally, modulating the expression of MRPS23 altered the sensitivity of the cells to CDK1 inhibitors. In conclusion, phosphorylation of MRPS23 by mitotic kinases might potentially be involved in the proliferation of breast cancer cells. Furthermore, MRPS23 can be targeted for sensitizing the breast cancer cells to CDK1 inhibitors.

MicroRNA expression profiling of endocrine sensitive and resistant breast cancer cell lines

BackgroundMicroRNAs (miRs) regulate gene expression through translation inhibition of target mRNAs. One of the most promising approaches for cancer therapy is through mimicking or antagonizing the action of miRs. In this report, we analyzed the miRnome profile of several human breast cancer cell lines to determine the influence of estrogen receptor (ER) silencing previously shown to result in epithelial to mesenchymal transition (EMT) and enhanced tumor invasion. MethodsMicroRNA extracted from MDA-MB-231 (de novo ER-) and ER-silenced (acquired ER-) pII and IM-26 or ER-expressing (YS1.2) siRNA transfected derivatives of MCF7 cells was deep sequenced on Illumina NextSeq500. Respective miRnomes were compared with edgeR package in R and Venny2.1 and target prediction performed with miRTarBase. Mimics and inhibitors of selected differentially expressed miRs associated with EMT mediators (miR-200c-3p targeting ZEB1, miR-449a targeting δ-catenin and miR-29a-3p) were transfected into pII cells and mRNA targets, as well as E-cadherin and keratin 19 (epithelial and mesenchymal markers respectively) were measured using taqman PCR. ResultsEach cell line expressed about 20% of the total known human miRnome; There was a high degree of similarity between the 3 tested ER-lines. Out of these expressed miRs, 50–60% were significantly differentially expressed between ER- and ER + lines. Transfection of miR-200c-3p mimic into pII cells down regulated ZEB1 and vimentin, and increased E-cadherin and keratin 19 with accompanying morphological changes, and reduced cell motility, reflecting a reversal back into an epithelial phenotype. On the other hand, transfecting pII with miR-449a inhibitor reduced cell invasion but did not induce EMT. Transfecting pII cell line with the mimic or inhibitor of miR-29a-3p showed no change in EMT markers or cell invasion suggesting that the EMT induced by loss of ER function can be reversed by blocking some but not just any random EMT-associated genes. ConclusionsThese data suggest that differences in miR expression can be exploited not only as mediators (using mimics) and targets (using miR antagonists) for general cancer therapies aimed at regulating either individual or multiple mRNAs, but also to re-sensitize endocrine resistant breast cancers by turning them back into a type that will be susceptible to endocrine agents.

The E3 Ligase TRIM4 Facilitates SET Ubiquitin‐Mediated Degradation to Enhance ER‐α Action in Breast Cancer

Estrogen receptor alpha (ER‐α) action is critical for hormone‐dependent breast cancer, and ER‐α dysregulation can lead to the emergence of resistance to endocrine therapy. Here, it is found that TRIM4 is downregulated in tamoxifen (TAM)‐resistant breast cancer cells, while the loss of TRIM4 is associated with an unfavorable prognosis. In vitro and in vivo experiments confirm that TRIM4 increased ER‐α expression and the sensitivity of breast cancer cells to TAM. Mechanistically, TRIM4 is found to target SET, and TRIM4‐SET interactions are mediated by the RING and B‐box domains of TRIM4 and the carboxyl terminus of SET. Moreover, it is determined that TRIM4 catalyzed the K48‐linked polyubiquitination of SET (K150 and K172), promoting its proteasomal degradation and disassociation from p53 and PP2A. Once released, p53 and PP2A are able to further promote ESR1 gene transcription and enhance mRNA stability. Moreover, univariate and multivariate Cox proportional hazards regression analyses confirm that TRIM4 expression is an independent predictor of overall survival and recurrence‐free survival outcomes in patients with ER‐α positive breast cancer. Taken together, the data highlights a previously undiscovered mechanism and suggest that TRIM4 is a valuable biomarker that can be analyzed to predict response to endocrine therapy in breast cancer patients.

Bovine serum albumin-gold nanoclusters protein corona stabilized polystyrene nanoparticles as dual-color fluorescent nanoprobes for breast cancer detection

Breast cancer is the most prevalent malignancy and the first leading cause of cancer-related mortality among the female population worldwide. Approaches for precise and reliable detection of breast cancer cells, particularly in the nascent state, are desperately needed for elevating the survival rate of patients bearing the breast tumor. In this work, we successfully performed the sensitive, precise, and reliable breast cancer cell detection using facilely fabricated bovine serum albumin-gold nanocluster (BSA-AuNCs) protein corona stabilized, epithelial cell adhesion molecule (EpCAM) aptamer linked fluorescent polystyrene nanoparticle (PS NP), termed as PS-BSA-AuNCs-Apt. The rapidly adsorbed BSA-AuNCs hard protein corona without complicated covalent conjugation not only imparted excellent colloidal stability to the PS nanoparticles, but also offered numerous active anchors for the targeted EpCAM aptamers to locate. With the remarkable aid of the aptamers specifically targeting the EpCAM-positive breast cancer cells, the PS-BSA-AuNCs-Apt emitted strong and photostable dual-color fluorescent signals for precise and reliable cancer cell detection by overcoming the false signals. The specific identification potency of the PS-BSA-AuNCs-Apt system was further verified by successfully detecting the xenografted breast tumor tissue. Notably, to the best of our knowledge, the protein corona formed nanoprobes was exploited for direct tumor cell and tissue detection with high efficacy for the first time, demonstrating their promising potential in clinical tumor detection.

The Role of Runx2 in Microtubule Acetylation in Bone Metastatic Breast Cancer Cells.

Simple SummaryBreast cancer is the most commonly diagnosed cancer type, making up a quarter of all cases among women. While modern-day research has shown tremendous progress in the development of treatment options, complications related to bone metastasis remain an obstacle that demands further attention. Cancer cells that migrate into the bone microenvironment show significant alterations in their metabolism and gene expression profiles. In this study, we set out to better understand the changes induced by Runx2 in bone-derived breast cancer cells. We identified key changes in the stability of the microtubule cytoskeleton of bone-derived cells expressing Runx2. These changes have implications on a variety of processes related to metabolism, cellular stress response, and response to common chemotherapies. These studies help to shed light on the changes that take place as a tumor begins to metastasize and to better predict which therapies will benefit patients with bone metastasis.Bone metastasis of breast cancer results in severe bone loss, fractures, and death. Crosstalk between breast cancer cells and bone resident cells promotes osteoclast activity and the release of growth factors from the bone matrix resulting in aggressive tumor growth and bone loss. We and others have shown that Runt-related transcription factor-2 (Runx2) promotes metastatic tumor growth-associated bone loss. Breast cancer cells also induce autophagy to survive metabolic stress at the metastatic site. Recently, we reported a Runx2-dependent increase in autophagy. In this study, to examine the underlying mechanisms of metastasis and tumor resistance to stress, we used a bone metastatic isogenic variant of breast cancer MDA-MB-231 cells isolated from a xenograft tumor mouse model of metastasis. Our results with immunofluorescence and biochemical approaches revealed that Runx2 promotes microtubule (MT) stability to facilitate autophagy. Stable MTs are critical for autophagosome trafficking and display increased acetylation at Lysine 40 of α-tubulin. Runx2 silencing decreases acetylated α-tubulin levels. The expression levels of HDAC6 and αTAT1, which serve to regulate the acetylation of α-tubulin, were not altered with Runx2 silencing. We found that HDAC6 interaction with α-tubulin is inhibited by Runt-related factor-2 (Runx2). We show that the expression of wild-type Runx2 can restore the acetylated polymer of MTs in Runx2 knockdown cells, while the C-terminal deletion mutant fails to rescue the polymer of MTs. Importantly, cellular stress, such as glucose starvation also increases the acetylation of α-tubulin. We found that the loss of Runx2 increases the sensitivity of breast cancer cells to MT-targeting agents. Overall, our results indicate a novel regulatory mechanism of microtubule acetylation and suggest that Runx2 and acetylated microtubules may serve as therapeutic targets for bone metastatic tumors.

A23187 May Improve Tamoxifen Sensitivity of Tamoxifen-Resistant Breast Cancer Cells

Giriş ve Amaç: Tamoksifen, ER α-pozitif meme kanserinin tedavisinde en yaygın kullanılan terapötik bir ajandır. Ancak hastaların büyük bir kısmında tamoksifen’e karşı direnç kazanımının oluşması; terapötik etkinliği sınırlamakta ve hastalarda sağ kalım oranını azaltmaktadır. Hücre hareketliliği, gen ifadesi regülasyonu gibi çok sayıda kritik rolü olan Ca+2 sinyal mekanizması karsinogenez ile ilişkili proliferasyon, migrasyon, anjiyogenez ve ilaç direnci gelişimi gibi süreçler üzerinde önemli rollere sahiptir. Çalışmalarımızda yüksek oranda Ca+2 seçiciliği olan ve endoplazmik retikulumdan Ca+2 çıkışına aracılık eden kalsiyum iyonofor A23187 (kalsimisin)’nin tamoksifene dirençli meme kanseri hücrelerinde proliferasyon ve tamoksifen direnci üzerine olan etkisinin araştırılması amaçlanmıştır.
 Gereç ve Yöntemler: A23187 veya Tamoksifen ile A23187 kombine uygulamasının Tamoksifene dirençli meme kanseri hücresi MCF-7/TAMR-1’de hücre proliferasyonu üzerine olan etkisini değerlendirmek amacıyla WST-1 temelli hücre proliferasyon analizleri gerçekleştirilmiştir. Ayrıca mikroskobik incelemeler yapılarak fotoğraflanmıştır. Bulgular: A21387’nin MCF-7/TAMR-1 hücreleri üzerindeki anti-proliferatif etkinliğe sahip olduğunu göstermiştir. A23187 ile tamoksifen’in kombine uygulaması ile hücrelerdeki tamoksifen direncini sınırlandırarak sinerjistik olarak hücrelerin proliferatif kapasitesini sınırladığı belirlenmiştir. 
 Sonuç: Bulgularımız, A23187 aracılı kalsiyum sinyalinin modülasyonunun meme kanseri hücrelerinde tamoksifen duyarlılığının ilerletilmesinde umut verici bir yaklaşım olabileceğini önermektedir.

Resistance of breast cancer cells to all-trans retinoic acid is associated with a decrease in the basal level of nuclear receptor <i>RARα</i> expression and induction of cytochrome <i>CYP26A1</i> and <i>CYP26B1</i> expression

Introduction. Retinoic acid (RA) is a key regulator of cell differentiation and a critical player in such systemic processes in the body as embryonic development, immune system cell maturation and functioning, tissue remodeling and several others. This compound displays an antitumor activity due to its ability to stimulate differentiation, induce apoptosis and inhibit proliferation of malignant cells. The rapid acquisition of resistance to RA and its analogues by solid tumor cells is one of the main problems limiting the widespread use of retinoids in the therapy of malignant neoplasms. The mechanisms of RA-resistance are still poorly understood.The study objective – assessment of the relationship between the basal expression level of the nuclear RARα receptor and the RA-induced expression of the cytochromes CYP26A1and CYP26B1 with the resistance of breast cancer cells to the action of all-trans-retinoic acid.Materials and methods. Cell lines were cultured, the sensitivity of breast cancer cells to the action of fully trans-retinoic acid, RNA isolation, reverse transcription reaction and real-time polymerase chain reaction were analyzed).Results. In present study, using an experimental model represented by 9 breast cancer cell lines with different level of sensitivity to RA, we showed that the expression of the RA nuclear receptor RARα, as well as the level of mRNA induction of CYP26A1 and CYP26B1 cytochromes in response to RA treatment correlate with RA-sensitivity.Conclusion. Thus, a decrease of RARα expression as well as the reduced ability to catabolize RA are factors associated with RA-resistance of breast cancer cells.

Investigating CENPW as a Novel Biomarker Correlated With the Development and Poor Prognosis of Breast Carcinoma.

Breast invasive carcinoma (BRCA) is a carcinoma with a fairly high incidence, and the therapeutic schedules are generally surgery and chemotherapy. However, chemotherapeutic drugs tend to produce serious toxic side effects, which lead to the cessation of treatment. Therefore, it is imperative to develop treatment strategies that are more effective and have fewer side effects at the genetic level. Centromeric protein W (CENPW) is an oncogene that plays an important part in nucleosome assembly. To date, no studies have reported the prognostic significance of CENPW in breast carcinoma. In this study, we verified that CENPW expression is up-regulated in breast carcinoma and positively associated with the level of immune cell infiltration. The clinicopathological characteristics further suggest that CENPW expression is correlated with a worse prognosis of breast carcinoma. Interestingly, the CENPW mutation contributes to the poor prognosis. Next, we discovered that the genes interacting with CENPW are mainly concentrated in the cell cycle pathway, and CENPW is co-expressed with CDCA7, which is also highly expressed in breast carcinoma and leads to a worse prognosis. Our subsequent studies verified that knockdown of CENPW significantly inhibits the proliferation and migration of breast carcinoma cells and promotes their apoptosis rate. Notably, inhibition of CEMPW sensitizes breast cancer cells to chemotherapeutic drugs that have been found to induce cell cycle arrest. In summary, these results provide extensive data and experimental evidence that CENPW can serve as a novel predictor of breast cancer and may act as a prospective therapeutic target.

SUFU suppresses ferroptosis sensitivity in breast cancer cells via Hippo/YAP pathway

SummaryFerroptosis is a new kind of regulated cell death that is characterized by highly iron-dependent lipid peroxidation. Cancer cells differ in their sensitivity to ferroptosis. Here we showed that the Suppressor of fused homolog (SUFU), a critical component in Hedgehog signaling, regulates ferroptosis sensitivity of breast cancer cells. Ectopic SUFU expression suppressed, whereas depletion of SUFU enhanced the sensitivity of breast cancer cells to RSL3-triggered ferroptosis through deregulation of ACSL4. Moreover, SUFU depletion promoted the activation of Yes-associated protein (YAP), thereby increasing the expression of ACSL4. Mechanistically, SUFU is associated with LATS1. Deletion of a region comprising residues 174–385 in SUFU disrupted SUFU binding to LATS1, thus abrogating SUFU-mediated downregulation of the YAP-ACSL4 axis and sensitivity to ferroptosis. Noteworthy, we showed that vincristine downregulated SUFU, thus increasing breast cancer cell sensitivity to RSL3 in vitro and in vivo. Together, our findings uncover SUFU as a novel regulator in ferroptosis sensitivity.

The Fractions of Eryngium thyrsoideum Extract Sensitize Breast Cancer Cells to Apoptosis

Background: Breast cancer (BC) is the most common cancer in women, and its prevalence has increased dramatically in recent years. Many treatments for BC have been proven, one of which is the utilization of natural products and herbal derivatives. Among natural plants, plants of the Apiaceae family like Eryngium have been studied, which entail antioxidant, antimicrobial, and most importantly, anti-cancer properties. Objectives: Considering the cytotoxical effects of different Eryngium species, it seems reasonable to evaluate the cytotoxic activity of E. thyrsoideum fractions on the BC, including MCF-7 and MDA-MB-231 cell lines. Methods: The shoots of E. thyrsoideum were extracted by Soxhlet apparatus with n-Hexane, methanol, and dichloromethane solvents. Methanolic extract (strong extract) on C18 Sep-Pak column with a mobile phase of methanol-water was fractionated. Then, the cytotoxicity of different fractions of the strong extract against normal and BC cells was evaluated for 24 and 48 hours using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. Induction of apoptosis was assessed by flow cytometry using staining of cells treated with Annexin V/PI. Results: The 80% fraction of the methanolic extract illustrated the strongest cytotoxic effectiveness among the others. This strong fraction specifically prohibited the MCF-7 and MDA-MB-231 growth with minimal effect on normal cells. The prohibition of cell growth had a time- and dose-dependent manner (P < 0.001). In addition, the flow cytometric analysis indicated that the strong fraction exerted its cytotoxic effects by inducing apoptosis on the cancer cell line. Conclusions: According to our results, due to effective secondary metabolites, 80% fraction of methanolic extract prohibited the growth of both types of BC cells by inducing apoptosis and had less toxicity on normal cells.

Abstract 3731: Epigenetic control of tumor cell killing by natural killer cells

Abstract Immunotherapies including the activation of endogenous immune cells have stimulated great interest for their anti-tumor functions. An initial focus has been on genetic lesions such as loss of MHC class I and class II molecules that facilitate tumor cell escape. More recently, epigenetic mechanisms such as the disruption of the SWI/SNF remodeling complex PBAF have been implicated in regulating tumor sensitivity to CD8 T cell-mediated killing. Here, I used an unbiased genetic screening approach to examine resistance and sensitivity of breast cancer cells to Natural Killer (NK) cells. Among expected hits including caspases, interferon receptors, and cell death executioners, the screen revealed a surprising role of alternate SWI/SNF complexes in controlling NK cell-mediated cytotoxicity that relate to interferon-γ and perforin/granzyme B, and may be pharmacologically reversible. Altogether, I will discuss a new epigenetic mechanism that affect NK cell-mediated cytotoxicity with a focus on SWI/SNF complexes. Citation Format: Joyce Pasion, Darko Barisic, Cem Meydan, Kong Y. Ng, Maria Tello Lafoz, Morgan Huse, Ari Melnick, Hans-Guido Wendel. Epigenetic control of tumor cell killing by natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3731.

Abstract 3285: Targeting STAT3 with novel liposome-incorporated antisense oligonucleotide technology enhances the efficacy of paclitaxel (taxol) and 5-fluorouracil (5-FU) in breast and ovarian cancer cells

Abstract Background: STAT3 is a transcription factor that regulates various tumorigenic processes, such as tumor proliferation, metastasis, and drug resistance. Its overexpression and aberrant activation characterize many cancers (including breast, lung, ovarian, liver and colon cancer). Metastasis and drug resistance are two main causes of patient mortality. Activation of the JAK-STAT3 and the FGFR1-STAT3 pathways in breast and ovarian cancer cells promotes tumor initiation, migration, and taxol resistance. STAT3 also contributes to epithelial-mesenchymal transition and promotes 5-FU resistance in colorectal cancer cells. Its role in numerous malignancies has made STAT3 an ideal cancer therapeutic target. BP1003 is a neutral DOPC liposome incorporated with a nuclease-resistant P-ethoxy antisense oligodeoxynucleotide targeting STAT3 mRNA. Here we determine the effects of BP1003 in combination with taxol and 5-FU on breast and ovarian cancer cells. Methods: Western blots determined the extent by which BP1003 reduced STAT3 protein levels. The effect of sequential taxol or 5-FU (5 h pre-treatment) and BP1003 treatments (96 h) on cell viability was analyzed with resazurin-based assays. Migration and colony formation assays were performed to assess the metastatic potential of treated cells. Cells pre-treated with BP1003 (96 h) and taxol (2 h) were placed in chemotaxis chambers and allowed to migrate for 5-18 h before quantification. Colony formation involved the treatment of a low number of cells with chemotherapeutic drugs for 24 h prior to the addition of BP1003 for 10-14 days. Tumor spheroids were used to mimic in vivo solid tumor growth. Three days after formation, spheroids were treated with chemotherapeutic drugs for 1 day before being treated with BP1003 for 3 additional days. Results: Treatment of BT549, SK-Br-3 and SK-Ov-3 cells with 250 μg/ml of BP1003 resulted in a 30%-50% decrease in STAT3 expression. Cell viability decreased by 20-35% with 200 μg/ml of BP1003 and by 45-60% in combination with taxol or 5-FU. BP1003 in combination with taxol also decreased the colony formation potential of BT549 cells by 20%. Migration of BT549 cells was reduced by 30% with 200 μg/ml BP1003 alone and by 50% in combination with taxol. The area of BT549 and SK-Ov-3 spheroids under BP1003 and taxol combination treatments was 40%-50% smaller than control spheroids and 20%-30% smaller than taxol mono-treatment. Conclusions: BP1003 efficiently reduces STAT3 expression and enhances the sensitivity of breast and ovarian cancer cells to taxol and 5-FU. These results are in line with previous work in which BP1003 + gemcitabine displayed enhanced anti-tumor activity in pancreatic ductal adenocarcinoma. Together these results strongly suggest that BP1003 combination therapy is a novel strategy for patients with advanced solid tumors. Citation Format: Maria Gagliardi, Ana Tari Ashizawa. Targeting STAT3 with novel liposome-incorporated antisense oligonucleotide technology enhances the efficacy of paclitaxel (taxol) and 5-fluorouracil (5-FU) in breast and ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3285.

Abstract 2327: Dysregulated TCA cycle is associated with endocrine therapy resistance in ER+ breast cancer cells

Abstract Estrogen receptor positive (ER+) breast cancer is the most common breast cancer subtype. Endocrine therapies, such as selective estrogen receptor modulators (SERM) and aromatase inhibitors (AI), along with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, are the current mainstays of treatment. Despite being initially effective, the majority of advanced ER+ breast cancers become resistant to the endocrine therapies. Therefore, it is important to understand the underlying molecular mechanisms that enable or promote resistance to endocrine therapies. Rapidly proliferating cells have a high energy demand and consequently consume greater quantities of glucose to fulfill their energy and anabolic demands to support cell growth. Using endocrine therapy sensitive and resistant breast cancer cell models we investigated the metabolic flux of glucose by growing the cells in the presence of the stable glucose isotope, UC13-Glucose and measured the C13-labelled metabolites of glycolysis and the TCA cycle. Glucose consumption was higher in endocrine therapy resistant MCF7/LCC9 (LCC9) cells compared with parental MCF7 (MCF7) and estrogen independent, endocrine therapy sensitive MCF7/LCC1 (LCC1) cells. The TCA cycle was noticeably impaired in LCC9 cells as the C-13 labelled citrate (m+4 and m+6) isotopomer was absent in these cells. In addition, C-13 labelled fumarate was very low in LCC9 cells with a concurrent increase in the succinate:fumarate ratio. These observations show that in LCC9 cells the TCA cycle is dysregulated at the succinate to fumarate conversion step, which is catalyzed by the succinate dehydrogenase (SDH) enzyme. SDH is a multimeric protein comprised of four different subunits and SDH assembly factor 2 (SDHAF2), which is a tumor suppressor gene. Further investigation revealed that SDHAF2 was under expressed in LCC9 cells compared with LCC1 cells, likely contributing to lower SDH activity. Intriguingly, SDH inhibition by dimethyl-malonate re-sensitized LCC9 cells to both Fulvestrant and 4-hydroxytamoxifen. Clinically, lower expression levels of SDHAF2 correlates with poor relapse free survival in ER+ breast cancer patients. In summary, our study suggests that accumulation of succinate driven by deficient SDH activity is functionally associated with endocrine therapy resistance in ER+ breast cancer cells. Citation Format: Surojeet Sengupta, Karla Andrade de Oliviera, Lu Jin, Robert Clarke. Dysregulated TCA cycle is associated with endocrine therapy resistance in ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2327.

Abstract 270: The novel control mechanism of the tubulin code and vesicular trafficking in breast cancer bone metastatic cells

Abstract Metastasis of breast cancer (BC) to bone results in severe bone loss, fractures, and death. The crosstalk between BC cells and bone resident cells dramatically increases osteoclast activity, resulting in the release of growth factors from the bone matrix that causes aggressive tumor growth and bone loss. A potentially important aspect of this process is vesicular trafficking on microtubules (MTs) which can affect the output of signaling pathways and secretory activity of metastatic bone cells. MTs are cytoskeletal filaments composed of heterodimers, α- and β-tubulin. The tubulin isotypes and their variety of post-translational modifications (PTMs) control the properties and functions of MT filaments, a concept known as the ‘tubulin code’. Recent studies show an emerging link between alterations of the tubulin code with poor prognosis of breast cancer. However, the regulation of the code in metastatic bone cells is currently unknown. Tubulin acetylation occurs via α-tubulin N-acetyl transferase-1 (αTAT1) and can be reversed by histone deacetylase-6 (HDAC6). MTs lacking acetylation lose flexural rigidity and are prone to breaks following repetitive bending during vesicular trafficking. We found that HDAC6 interaction with α-tubulin is inhibited by a Runt-related factor (Runx2). Our biochemical, mass spectrometry and IPs analyses of MTs revealed that loss of Runx2 can reduce (i) acetylation and stability of MTs, (ii) interaction of HSP90 with α-tubulin, and (iii) levels of β 2a-tubulin. Our studies with Runx2 mutants indicate that the C-terminal of Runx2 serves a scaffolding function by interacting with MTs and HDAC6. Confocal microscopy revealed reduced puncta and altered distribution of endosomal vesicles and autophagosomes with Runx2 silencing. As MT targeting agents are often used as chemotherapeutics, we found that loss of Runx2 sensitizes breast cancer cells to docetaxel and vinblastine and reduces the secretion of IL-6. We found MDA-MB-231 cells metastasized to the bone show significant differences in microtubule isotype expression, with 10 of 19 studied showing 1.3 to 4.1 fold increases. Further analysis of whether Runx2 knockdown affects isotype expression, we found the majority of the 19 studied remain unchanged. Immunohistochemistry for Ac-α-Tub levels in bone metastatic patient samples shows significantly more Ac-α-Tub positive cells in metastatic bone tumors than primary tumors. These findings suggest a novel control mechanism of MTs stability via Runx2-HDAC6 interactions that can impact trafficking and cellular activity. Our results indicate that inhibition of Runx2 may sensitize metastatic tumors to MT targeting agents, and Runx2/HDAC6/Ac-α-Tub levels may serve as markers for metastatic tumors to help stratify patients for optimal treatment for metastatic bone disease. Citation Format: Shreya Patel, Marcus Winogradzki, Ahmad Othman, Waddell Holmes, Jitesh Pratap. The novel control mechanism of the tubulin code and vesicular trafficking in breast cancer bone metastatic cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 270.

LINC00094/miR-19a-3p/CYP19A1 axis affects the sensitivity of ER positive breast cancer cells to Letrozole through EMT pathway.

The endocrine therapy resistance of breast cancer is the difficulty and challenge to be urgently solved in the current treatment. In this study, we examined the effects of noncoding RNA LINC00094 and miR-19a-3p on breast cancer in vivo and in vitro by RT-QPCR, Western Blot, luciferase assay, immunofluorescence and drug sensitivity tests. The plasma level of CYP19A1 in patients with breast cancer resistance was lower than that in drug sensitive patients. Compared with normal subjects, miR-19a-3p was highly expressed in plasma of patients with breast cancer. miR-19a-3p is highly expressed in estrogen receptor positive breast cancer cells. The expression of miR-19a-3p promoted the migration and EMT of breast cancer cells and reduced the sensitivity of breast cancer to Letrozole. LINC00094 sponge adsorbed miR-19a-3p. LINC00094 promotes the expression of CYP19A1, the target gene of miR-19a-3p, and inhibits the EMT process of breast cancer, ultimately promoting the sensitivity of ER-positive breast cancer cells to Letrozole. This study found a new mechanism of Letrozole sensitivity in ER positive breast cancer.

A deep learning approach to discover cyclin-dependent kinases 12 (CDK12) inhibitors in breast cancer.

e15086 Background: Although anti-HER2 tyrosine kinase inhibitors (TKIs) have significantly prolonged survival and improved prognosis in HER2-positive breast cancer patients, resistance is a constant obstacle leading to TKIs treatment failure and tumor progression. Our previous research shows dual inhibition of HER2/CDK12 will prominently benefit the outcomes of patients with HER2-positive breast cancer by sensitizing the tumors to anti-HER2 TKIs treatment. Unfortunately, there is no high selective CDK12 inhibitor in clinical. This study sought to accelerate the CDK12 inhibitor discovery by a deep learning approach. Methods: We developed a large-scale self-supervised graph neural network to learn 11 million unlabelled compounds for molecular representation. And the model was trained with 233,823 kinase domain sequences by drug-target interaction datasets from the BindingDB database. Then, this transformer architecture was used to predict potential CDK12 inhibitors. The candidate hits compounds with diverse skeletons were divided into 50 clusters by clustering analysis. The representative hits with high transform scores and rational binding mode in each cluster were detected in the homogeneous time-resolved fluorescence CDK12 kinase assay. Candidate clusters with half-maximal inhibitory concentrations (IC50) lower than 200 nM were selected into expanded validation and kinase selective panel detection. xCELLigence RTCA system was adopted to monitor cell growth and sensitivity of breast cancer cells to CDK12 inhibitors in a real-time manner. Results: Our predictive transfer learning model yielded satisfactory predictions on various targets including CDK12. We screened a total of 4,527,236 compounds and recommended 50 clusters of potential CDK12 inhibitors for further enzyme activity detection. The IC50 of seven cluster representative compounds is lower than 10 uM. Expanded screening of 500 compounds in these clusters discovered a lot of hits with highly CDK12 selective inhibition. Half of the new compounds are showed promising proliferation inhibition in breast cancer cell lines. Conclusions: Our study provides a highly efficient and end-to-end deep learning approach to discover highly selective CDK12 inhibitors in breast cancer.