A Simple and Cost-Effective Method for Generating Spheroids From Triple-Negative Breast Cancer Cell Line (MDA-MB-231)

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

Background: Ovarian cancer is the leading cause of death from gynecologic malignancies in developed countries. High-grade serous ovarian cancer (HGS-OvCa) is often initially sensitive to platinum-based therapy, but relapse rates remain high. The TCGA recently found that HGS-OvCas have a gene expression and mutational profile similar to that of triple negative breast cancer (TNBC). Previously, our group demonstrated that dexamethasone treatment decreased chemotherapy-induced tumor cell apoptosis in TNBC and HGS-OvCa cell lines. We have also shown that glucocorticoid receptor (GR) activation induces expression of anti-apoptotic genes SGK1 and MKP1/DUSP1 in both HGS-OvCa and TNBC cell lines and in primary human ovarian and TNBC tumors. Methods: We examined glucocorticoid receptor (GR), estrogen receptor (ER), and progesterone receptor (PR) expression in a panel of HGS-OvCa cell lines by Western analysis and qRT-PCR. We evaluated GR target genes SGK1, MKP1/ DUSP1, and GILZ to determine if there is a difference in expression of the anti-apoptotic genes when exposed to glucocorticoids versus glucocorticoid antagonist. We also performed apoptosis assays with and without mifepristone, glucocorticoid and/or chemotherapy treatment using IncuCyte™ live-cell imaging technology in order to measure the effect of GR modulation in chemotherapy sensitivity. In addition, we examined human tissue microarrays of various subtypes of epithelial ovarian carcinoma using immunohistochemical stain (IHC) to examine the tumors for ER, PR, and GR expression. Results: HGS-OvCa cell lines (including CAOV3, HeyA8, SKOV3, Monty-1) all had detectable GR expression; HeyA8, SKOV3, and Monty-1 cell lines expressed very low levels of ER-alpha while all other HGS-OvCa cell lines did not express any detectable ER-alpha. Furthermore, none of the HGS-OvCa cell lines tested expressed PR. Similar findings of GR and PR expression were also demonstrated in the primary ovarian cancer tissue microarrays. Known GR target genes SGK1, MKP1, and GILZ were upregulated in the presence of the glucocorticoid agonist dexamethasone and the addition of mifepristone inhibited this response. Apoptosis assays revealed that GR activation significantly inhibited carboplatin/gemcitabine-induced apoptosis in HGS-OvCa cell lines and that mifepristone could inhibit this cell survival effect, presumably through GR antagonism. Conclusions: These results suggest that treatment with mifepristone, a GR antagonist, reverses GR-mediated cell survival signaling in HGS-OvCa and increases chemotherapy-induced tumor cell death. This preliminary data may suggest that the glucocorticoid receptor may potentially be a good target for future therapeutics. To further investigate the role of GR activity in HGS-OvCas in vivo, we are currently performing xenograft experiments with chemotherapy +/- mifepristone treatment and exploring phase I clinical trials. Citation Format: Erica M. Stringer, Maxell N. Skor, Lei Zhao, Katja Gwin, Ernst Lengyel, Gini F. Fleming, Suzanne D. Conzen. The role of the glucocorticoid receptor (GR) in inhibiting chemotherapy-induced apoptosis in high-grade serous ovarian carcinoma (HGS-OvCa). [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B32.

Breast cancer is one of the most commonly diagnosed cancers and the leading cause of cancer-related death among women. Triple negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. TNBC shows a high capacity for early metastasis and leads to worse clinical outcomes than other breast cancer subtypes due to the lack of specific therapeutic targets. We are looking to develop cancer-specific therapeutic targets for TNBC. Many types of cancer cells including TNBC cells rely on glutamine as carbon and nitrogen source to fuel unchecked growth. We screened for key genes in regulating glutamine metabolism in a panel of breast cancer cell lines. This screen identified the mRNA and protein levels of solute carrier family 38 member 3 (SLC38A3), a glutamine transporter, to be upregulated in human breast cancer cell lines, especially in TNBC cell lines. The TCGA breast cancer patient database also showed that SLC38A3 mRNA is overexpressed in invasive ductal breast carcinoma tissues, and it is even higher in TNBC relative to other breast cancer subtypes. To test the biological role of SLC38A3 protein in TNBC cells, we performed loss-of-function experiments in multiple TNBC cell lines. Silencing of SLC38A3 decreased cellular glutamate, glutamine and alanine levels. Silencing of SLC38A3 also activated apoptosis, and suppressed cell viability, migration and invasion in several TNBC cell lines. Interestingly, silencing of SLC38A3 increased the activity of glycogen synthase kinase 3-β (GSK3β) which promoted the degradation of β-catenin, leading to the decrease of the mRNA levels of epithelial-to-mesenchymal-transition (EMT)-associated transcription factors in TNBC cell lines. In summary, we showed that SLC38A3 is overexpressed in TNBC and promotes breast cancer migration and invasion via GSK3β/β-catenin/EMT pathway, which could be a novel therapeutic target for breast cancer. Citation Format: Zheqiong Tan, Caitlin M. Tressler, Kanchan Sonkar, Kristine Glunde. Glutamine transporter SLC38A3 promotes breast cancer migration via GSK3beta/beta-catenin/EMT pathway [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 LB207.

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

Many tumor cells are dependent on glutamine (Gln) and Gln-derived metabolites to meet bioenergetic and biosynthetic demands. A key cellular reaction in the utilization of Gln is its deamidation by the enzyme glutaminase to yield glutamate (Glu). We have developed a series of potent and selective small molecule glutaminase inhibitors for evaluation as novel cancer therapeutics. These inhibitors exhibit 5-20 nM potency against the broadly-expressed form of glutaminase (GLS) with minimal activity against the liver form of the enzyme (GLS2). Expression analysis (see below) has identified triple-negative breast cancer (TNBC) as a potential clinical target population for GLS inhibitors. TNBC is a poor prognosis breast cancer subtype lacking estrogen receptor (ER), progesterone receptor (PR) and the growth factor receptor Her2. TNBC is insensitive to approved targeted therapies (ER antagonists and anti-Her2 agents) and is currently treated with conventional cytotoxic drugs. Therefore, TNBC represents a critical unmet medical need for which new targeted therapeutics are urgently needed. Analysis of a primary breast tumor mRNA expression dataset (The Cancer Genome Atlas; n=756) revealed that low ER, PR, and Her2 expression is associated with high GLS expression and low expression of both GLS2 and glutamine synthetase, an enzyme that opposes the action of glutaminase by synthesizing Gln from Glu. This expression pattern, together with published work on the Gln dependence of breast tumor cell lines [Kung et al. (2011) PLOS Genet 7:e1002229] suggests that TNBC may be particularly dependent on GLS. To test this hypothesis, we evaluated the anti-tumor activity of our GLS inhibitors on a panel of breast tumor-derived cell lines (n>25) that included a mixture of TNBC and ER-positive subtypes. The TNBC subtype displayed the greatest sensitivity to GLS inhibitor treatment (IC50s ranging from 5-100 nM) and this sensitivity correlated with a dependence on extracellular Gln for cell growth. In the TNBC cell line MDA-MB-231 the antiproliferative effect of the GLS inhibitor was associated with a dose-dependent accumulation of Gln and depletion of Glu. Additionally, GLS inhibition showed additive in vitro activity in combination with paclitaxel, a standard-of-care treatment in TNBC. In a mouse orthotopic tumor xenograft model with MDA-MB-231 cells implanted in the mammary fat pad, oral delivery of a GLS inhibitor caused an accumulation of tumor Gln, a reduction in tumor Glu, and enhanced the anti-tumor efficacy of the paclitaxel. Experiments aimed at expanding this observation with other TNBC xenograft models are in progress. Overall, these results demonstrate that a selective inhibitor of GLS, either as a single agent or in combination with standard-of-care chemotherapeutics, may be effective as a targeted therapeutic in TNBC. Citation Format: Susan Demo, Tania Chernov-Rogan, Matthew Gross, Julie Janes, Raja Kawas, Evan Lewis, Francesco Parlati, Hector Rodriguez, Mirna Rodriguez, Jinfu Yang, Frances Zhao, Adam Richardson, Mark K. Bennett. Preclinical antitumor activity of novel small molecule glutaminase inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5604. doi:10.1158/1538-7445.AM2013-5604

The estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2, also known as ErbB-2 or c-neu) are the major biomarkers that define the classification and treatment options for patients with breast cancers. ER and PR positive breast cancers are more likely to respond to hormonal therapy. HER2 positive breast cancers are more likely to respond to trastuzumab targeted therapy. On the other end, triple negative breast cancers (TNBC) which lack ER and PR and do not overexpress HER2 do not respond well to hormonal therapy or HER2 targeted therapy and are associated with poor prognosis. Once patients with TNBC develop chemoresistance or relapse, there is no additional treatment option available. The key for developing novel therapeutics in TNBC is to elucidate the exact mechanisms by which these receptors undergo silencing, especially ER and PR. MicroRNAs (miRNAs), a class of small noncoding RNAs, have been shown to be important gene regulators in all biological processes including breast carcinogenesis and progression. Through an in silico analysis of the latest miRNAs database with four different miRNA algorithms, we have identified 11 putative miRNAs that target all 3′-untranslated regions (3’-UTRs) of the ER, PR and HER2 genes. miR-495 was one of the putative miRNAs that has been verified to be a significant regulator of ER and PR. Ectopic expression of miR-495 mimics in BT474 and MDA-MB-361 breast cancer cells greatly reduced ER and PR protein expression, but not HER2 expression. Enforced expression of a miR-495 inhibitor or antigomiR-495 in MDA-MB-361 breast cancer cells significantly elevated ER and PR expression. More importantly, miR-495 markedly inhibited the luciferase activities of both ER and PR reporters that contained the wild-type 3’-UTRs of ER and PR. miR-495 could not, however, affect the luciferase activities of the mutant ER and PR reporters that had been deleted the seed sequences in the 3’-UTRs of ER and PR, indicating that ER and PR are direct downstream targets of miR-495. miR-495 expression in primary breast cancers was assessed by in situ hybridization on a tissue microarray that containing 97 cases of randomly selected breast cancers. miR-495 levels were upregulated in thirteen breast cancers (13.4%). Among these 13 cases, 6 were TNBCs (46.2%), 2 were ER/PR low expressing breast cancers and 1 was ER/PR negative HER2 positive breast cancer. Therefore, miR-495 is a novel negative regulator of ER and PR and is upregulated in primary TNBC. Selective inhibition of miR-495 expression in TNBC with miR-495 inhibitors or antigomiR-495 may restore ER and PR expression. Citation Format: Xiao-Feng Le, Hui Ling, Maggie Mao, Xinna Zhang, Shu Zhang, George A. Calin, Yun Wu, Robert C. Bast. miR-495 functions as a novel regulator of the estrogen and progesterone receptorsin human breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3055. doi:10.1158/1538-7445.AM2013-3055

Epigenetic Regulation of Cancer Stem Cell Genes in Triple-Negative Breast Cancer

Background: Triple negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) overexpression. This aggressive form of breast cancer carries a higher risk of recurrence compared to other subtypes, presenting a significant clinical challenge. Although chemotherapy is commonly used as the primary treatment for a substantial number of TNBC patients, it is often accompanied by drug resistance and frequent tumor recurrence. Urgent efforts are needed to identify novel molecular targets specific to TNBC and develop effective therapies to combat this aggressive disease. The ultimate objective is to discover treatments capable of overcoming drug resistance and improving the overall survival of patients with TNBC. Methods: Immunohistochemistry was performed to examine the expression of HER3 in TNBC samples. Western blots were used to assess protein expression and activation. Cell proliferation and viability were determined by cell growth (MTS) assays. TCGA databases were analyzed to correlate HER3 mRNA expression with the clinical outcomes of TNBC patients. Specific shRNA was used to knockdown HER3 expression. LIVE/DEAD Cell Imaging was to detect live and dead cells. Orthotopic tumor models were established in nude mice to determine the capability of TNBC cells forming tumors and to test if our newly developed anti-HER3 monoclonal antibody (mAb) 4A7 could potentiate the antitumor activity of paclitaxel in vivo. Results: The chemo-resistant subline HCC1806-TR was obtained by subjecting TNBC HCC1806 cells to prolonged treatment with paclitaxel. In comparison to the parental cell line HCC1806-P, HCC1806-TR exhibited enhanced HER3 expression and activation. It is noteworthy that approximately half of the tested TNBC specimens and cell lines displayed elevated expression of HER3. Analyzing TCGA databases revealed that TNBC patients with high levels of HER3 mRNA expression in their tumors had significantly poorer overall survival (OS) and relapse-free survival (RFS) compared to those with low HER3 expression. Specifically knockdown of HER3 markedly inhibited TNBC cell proliferation and mammosphere formation in vitro and tumor growth in vivo. Blockade of HER3 signaling with our mAb (4A7) in combination with paclitaxel exerted significant antitumor activity against TNBC in vitro and in vivo. Conclusions: Our data demonstrate that increased HER3 is an effective therapeutic target for TNBC and our anti-HER3 mAb (4A7) may enhance the efficacy of paclitaxel in TNBC. Keywords: HER3, Anbibody, Chemotherapy, Triple Negative Breast Cancer Citation Format: Hui Lyu, Sanbao Ruan, Congcong Tan, Ann Thor, Bolin Liu. Targeting of HER3 potentiates the antitumor activity of paclitaxel against triple negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-24-08.

11101 Background: Ovarian cancer is the leading cause of death from gynecologic malignancies. High-grade serous ovarian cancer (HGS-OvCa) is often initially sensitive to platinum-based therapy, but relapse rates remain high. The TCGA recently found that HGS-OvCas have a gene expression and mutational profile similar to that of triple negative breast cancer (TNBC). Previously, our group demonstrated that dexamethasone treatment decreased chemotherapy-induced tumor cell apoptosis in TNBC and HGS-OvCa cell lines. We have also shown that glucocorticoid receptor (GR) activation induces expression of anti-apoptotic genes SGK1 and MKP1/DUSP1 in both HGS-OvCa and TNBC cell lines and in primary human ovarian and TNBC tumors. Methods: We examined glucocorticoid receptor (GR), estrogen receptor (ER), and progesterone receptor (PR) expression in a panel of HGS-OvCa cell lines by Western analysis and qRT-PCR. We also performed apoptosis assays with and without mifepristone, glucocorticoid and/or chemotherapy treatment using IncuCyte live-cell imaging technology in order to measure the effect of GR modulation of chemotherapy sensitivity. Results: HGS-OvCa cell lines (including CAOV3, HeyA8, SKOV3, Monty-1) all had detectable GR expression; HeyA8, SKOV3, and Monty-1 cell lines expressed very low levels of ER-alpha while all other HGS-OvCa cell lines did not express any detectable ER-alpha. Furthermore, none of the HGS-OvCa cell lines tested expressed PR.Apoptosis assays revealed that GR activation significantly inhibited gemcitabine/carboplatin-induced apoptosis in HGS-OvCa cell lines and that mifepristone could reverse this cell survival effect, presumably through GR antagonism. Conclusions: These results suggest that treatment with mifepristone, a GR antagonist, reverses GR-mediated cell survival signaling in HGS-OvCa and increases chemotherapy-induced tumor cell death. To further investigate the role of GR activity in HGS-OvCa, we are currently performing xenograft experiments with chemotherapy +/- mifepristone treatment.

To investigate the expression of estrogen receptor (ER), progesterone receptor (PR), and human epithelial growth factor receptor 2 (HER2) in breast cancer, and to explore their correlation with the age of patient, and size, clinic stage, and lymph node metastasis of the tumor. The data of 910 breast cancer, 89.4% of invasive ductal carcinoma, 1.7% of invasive lobular carcinoma, 44 cases 5% of ductal carcinoma in situ, and 4.9% of other pathologic types, 29.9% being less than 2 cm, 45.6% being 2-5 cm, and 24.5% bigger than 5 cm in size, 54.2% without metastasis in lymph node, 25.5% with metastasis in 1-3 lymph nodes, and 20.3% with metastasis in more than 3 lymph nodes respectively, were analyzed retrospectively. Immunohistochemistry was used to detect the expression of ER, PR, and HER2. The ER negative expression rate was 33.0%, and PR negative expression rate was 27.4%, and HER2 overexpression rate was 20.3%. The possibility of lymph node metastasis decreased along with the increase of age (P < 0.001). Tumor size was negatively correlated with the expression of ER and PR (both P < 0.001), and positively correlated with the expression of HER2 (P < 0.001). There was no significant difference between the situation of lymph node metastasis and the expression of ER, PR and HER2 in primary tumors. As good prognostic markers of breast invasive ductal cancer, ER and PR are negatively correlated with the HER2 expression, as a worse prognostic marker. ER/PR positive or HER2 negative tumors are morel likely to be diagnosed at earlier stages.

Breast cancer is the most common cancer among women, with approximately 2.3 million new cases globally. Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by the lack of estrogen receptor (ER), progesterone receptor (PR), and HER2 expression, making it unresponsive to traditional therapies. Eukaryotic Elongation Factor 2 Kinase (eEF2K) is overexpressed in TNBC, promoting cell survival by inhibiting apoptosis through phosphorylation of eEF2. Recently, eEF2K has been targeted for cancer therapy, and siRNA-based gene therapy has emerged as an effective approach to silence overexpressed genes. However, siRNA delivery is challenging due to its instability and susceptibility to degradation. In this study, we developed a novel hybrid nanoparticle (HNP) using a Layer-by-Layer (LbL) method for siRNA delivery targeting eEF2K in TNBC. The HNPs consist of a silver nanoparticle (AgNP) core, coated with poly (allylamine hydrochloride) (PAH) and poly(styrene sulfonate) (PSS), and loaded with eEF2K-siRNA and quercetin (QU), a chemotherapeutic agent, in separate layers. The nanoparticles also incorporated 4-ATP molecules for Raman traceability. In vitro experiments on TNBC cell lines (MDA-MB-231, BT-549, 4T1) showed that the combination therapy of eEF2K-siRNA and QU reduced cell viability, inhibited colony formation, and suppressed cell migration. At high 120 nM of siRNA concentration, 3D spheroid disintegration, activation of apoptotic pathways, and eventual necrotic cell death were observed. The results demonstrate that the developed HNPs are non-toxic, effective, and offer potential as a theranostic platform for TNBC treatment.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1007/s13346-025-01860-6.

To assess prognostic significance of progesterone receptors (PR) and estrogen receptors (ER) expression in the tissue microarray (TMA) technique for disease free survival (DFS) and overall survival (OS) in endometrioid endometrial cancer (EEC). The study included 151 consecutive patients, aged 37-86 years (62.80 +/- 9.99), with the EEC in stages I-III (FIGO), treated surgically at the Pirogow Memorial Hospital of Lodz between 2000 and 2007. Afterwards, they were subsequently treated and examined at the Regional Cancer Center, Copernicus Memorial Hospital of Lodz. Tissue cores 2 mm in size, in duplicate, were taken from the formalin-fixed and paraffin-embedded tissue donor blocks from surgery and constructed into the TMA recipient blocks. Using TMAs, the expression of PR and ER was examined and presented as Total Score (TS). The TS was determined by adding the intensity and marker distribution scores in a given case. The relationship between PR and ER expression, DFS and OS was examined. DFS was defined as the period from primary surgery until relapse. OS was defined as the period from primary surgery until the end of the follow-up (60 months) or until the death of the patient. The study was approved by the Ethics Committee of the Medical University of Lodz (RNN/82/11/KE). Lack of the PR and ER expression was found in 46 cases (30.46%) and 67 cases (44.37%), respectively. The expression of the PR and ER was weak in 24 cases (15.89%) and 22 cases (14.57%), respectively. Strong PR and ER expression was found in 81 patients (53.65%) and 62 patients (41.06%), respectively. Follow-up after surgery varied from 3 to 60 months (50.95 +/- 16.36). In 30 patients (19.87%) relapse was diagnosed 1-54 months (22.17 +/- 15.59) after surgery. During follow-ups, 29 patients (19.21%) died. In univariate analysis better DFS was related to the presence of PR (p = 0.010), higher TS of PR (HR = 0.81; 95% CI 0.71-0.94), the presence of ER (p = 0.001) and higher TS of ER (HR = 0.88; 95% CI 0.78-0.99). DFS differed significantly between the groups: without PR and ER expression (A), with presence of the PR but not ER expression (B), with the ER but not PR expression (C) and with the PR and ER expression (D) (p = 0.004). In univariate analysis OS was not related to PR expression (p = 0.110), TS of PR (HR = 0.89; 95% CI 0.80-1.02) and ER expression (p = 0.070). TS of ER was connected to better OS (HR = 0.83; 95% CI 0.72-0.96). The OS differed between groups A, B, C and D (p = 0.006). In multivariate analysis variants of PR/ER expression influenced the DFS (p = 0.039) and OS (p = 0.016). The expression of the PR and ER can significantly affect therapeutic decisions in selected patients with EEC. In EEC, common assessment of PR and ER expression is of higher prognostic value, than compared to single evaluation of PR and ER receptors.

Does triple-negative phenotype accurately identify basal-like tumour? An immunohistochemical analysis based on 143 ‘triple-negative’ breast cancers

10526 Background: The association between breast cancer characteristics and survival with estrogen receptor (ER) and progesterone receptor (PR) expression has been primarily studied via binomial categories, ER-positive and ER-negative. In order to better characterize germline genetic influences on these markers, we investigated their IHC expression semi-quantitatively in cancer predisposition germline pathogenic variant (PV) carriers of the following genes: BRCA1, BRCA2, PALB2, TP53, PTEN, CDH1, ATM, CHEK2, and Lynch syndrome genes. The HER2 expression was also analyzed. Methods: We conducted a retrospective chart review of patients with germline panel genetic testing for cancer predisposition genes at Moffitt Cancer Center’s GeneHome clinic. Inclusion criteria included 1) women ≥18 years old, 2) breast cancer diagnosis, 3) cancer predisposition germline panel genetic test results, 4) available ER and PR expression levels, and 5) available HER expression and/or amplification status. ER, PR, and HER2 status were compared between PV carriers and non-PV carriers via Mann-Whitney U at p&gt;0.05. Results: A total of 847 cases were reviewed for the study. Among 658 patients with a breast cancer diagnosis and complete ER PR data, 365 cases (55.5%) were non-PV carriers and 293 cases (44.5%) carried a PV in at least one of the genes listed above. Among 635 cases with available HER2 expression/amplification status, 355 (55.9%) cases were non-PV carriers and 288 (45.4%) cases were PV-carriers. When compared with non-PV carrier controls, BRCA1 PV carriers’ breast tumors had significantly lower ER and/or PR expression. Further, BRCA2 and TP53 PV tumors also displayed moderately lower ER expression. Contrarily, CHEK2 tumors displayed higher ER and PR expression compared to controls. Further, BRCA1 and BRCA2 PV carriers were more likely to have HER2- breast cancers. Conclusions: Differences in ER, PR, HER2 expression levels were observed in germline PV carrier breast cancers, signaling differential impacts by germline PVs on the tumor evolution process. It is likely that tumor differences in PV carriers influence responses to therapies, including hormone therapy, anti-HER2 therapy, and subsequent survival.[Table: see text]

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor, and HER2 expression. This lack of targetable receptors makes TNBC particularly challenging to treat. Identifying therapeutic targets which can be targeted to prevent TNBC recurrence is crucial for developing better treatments. By studying mechanisms driving recurrence we hypothesized that post-therapy changes in cancer cell epigenetics contribute to recurrence and that using epigenetic drugs to inhibit these changes could reduce the likelihood of relapse and improve patient outcomes. Thus, the aim of this study was to identify novel epigenetic regulators of tumor recurrence in TNBC. Using TNBC cell line MDA-MB-231, we generated a model of recurrence using Doxorubicin (Doxo). We performed a drug screen using commercially available epigenetic inhibitors in vitro and in vivo to identify novel epigenetic regulators of tumor recurrence. Cell count (in vitro) and tumor volumes (in vivo) were used to determine the anti-tumor effects. Weight loss was used as an index of toxicity in mice. Supporting our hypothesis, we observed notable differences in the levels of various epigenetic regulators before and after Doxo treatment and during recurrence. Additionally, we discovered that certain epigenetic drugs could prevent recurrence in culture, but only when administered sequentially following Doxo exposure, and not when used in prepriming or in combination. Sequential administration of the KAT inhibitor MG149 effectively controlled tumor growth in NSG mice with MDA-MB-231 and PDX1 tumors. MG149 was also found to enhance the effectiveness of other standard chemotherapy drugs, including paclitaxel and cyclophosphamide. In vitro and in vivo studies showed that sequential use of MG149 with Doxo increases DNA damage (gamma H2AX) and apoptosis (annexinV/7AAD) and significantly reduces the expression of cell cycle inhibitors p21, p27, and p53. These findings suggest a model where suppression of cell cycle inhibitors and persistent DNA damage post-therapy exposure with MG149 leads to increased apoptosis, reduced recurrence from therapy, and enhanced tumor growth control. Our findings indicate that new epigenetic states are formed following chemotherapy exposure, which can be therapeutically targeted to prevent recurrence in TNBC. Citation Format: Christiana O. Appiah, Manjulata Singh, Christiane Morecock, Aashka Shah, Abigail Andrews, Nasser Alabdulaly, Akash Jagdeesh, Sidney Umana-Guardado, Jonathan Yun, Laura Graham, Carson Walker, Madelyn Lorenz, Shengzhe Shang, Anthony Faber, Andrew Souers, Dipankar Bandyopadhyay, Steve Grant, Gordon Ginder, Jennifer Kobilinski, Harry D. Bear, Joshua C. Harrell, Joseph W. Landry. Lysine acetyltransferase inhibitor MG149 reduces tumor recurrence in triple-negative breast cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2728.