HER2 In Breast Cancer Cells Research Articles

Anticancer activity of the ethylacetate fraction of Vernonia amygdalina Delile towards overexpression of HER-2 breast cancer cell lines

Vernonia amygdalina Delile, coming from the Asteraceae tribe, contains active compounds that can treat breast cancer. This study examines the anticancer activity of Vernonia amygdalina Delile leaves, an active fraction of MCF-7/HER-2 breast cancer cells. Thin-layer chromatography determines the phytochemical screening. Cytotoxic and proliferation analyses were determined using the MTT method for the MCF-7/HER-2 breast cancer cell line. The cell cycle and apoptosis profiles were examined using flow cytometry. The results can be summarized in this study: Vernonia amygdalina Delile fraction contains a flavonoid with great potential for pharmacological activities, for instance, inhibiting the growth of cancer cells. The ethyl acetate fraction was more potent and cytotoxic on MCF-7/HER-2 cells (IC50 = 66 μg/mL) than extract ethanol (IC50 = 130 μg/mL). The ethylacetate fraction of Vernonia amygdalina Delile has been proven to inhibit cell proliferation by decreasing cell viability with an IC50 of 66 μg/mL concentration incubated for 24, 48, and 72 hrs with cell viability values of 64.46%, 61.67%, and 53.89%, respectively, compared to the ethanol extract IC50 of 130 μg/mL concentration with cell viability values of 56.0%, 50.19%, and 58.67%, respectively, which induced apoptosis and inhibited the cell cycle in the G2-M phase. To conclude, the ethyl acetate fraction of Vernonia amygdalina Delile can be used as an anticancer against the MCF-7/HER-2 cell line with an IC50 of 66 μg/mL, inhibiting cell proliferation and the cell cycle and inducing apoptosis activity in the MCF-7/HER-2 cell.

Abstract PO5-06-01: Loss of hormone receptor expression in breast cancer is associated with increased brain tropism and accelerated progression of leptomeningeal disease

Abstract The four breast cancer subtypes—luminal A, luminal B, HER2-overexpressing, and triple-negative breast cancer (TNBC) – are classified based on gene expression profiles of hormone receptors (HRs) (estrogen receptor (ER) and progesterone receptor (PR)) and human epidermal growth factor receptor 2 (HER2/ERBB2). Negative hormone receptor status is known to be associated with more aggressive breast cancer. Compared to other breast cancer subtypes, the HR-negative TNBC and HER2-overexpressing subtypes are characterized by high rates of recurrence and a notable increase in brain metastases and in particular, leptomeningeal disease (LMD). LMD – the most aggressive form of central nervous system (CNS) metastasis, in which cancer cells infiltrate leptomeninges of the brain and spine – carries a dismal prognosis due to a rapid progression, poor detection, and no effective treatment. Several studies that investigated the link between HR status and LMD suggest that HR-negative breast cancers have a shorter time to LMD diagnosis. This shortened timeline is consistent with higher migratory capabilities reported for TNBC and HER2 breast cancer cells. However, as it is known that breast cancers exhibit subtype-specific metastatic tropism (i.e., luminal subtypes tend to metastasize to bone and liver, while TNBCs metastasize to the CNS), the longer time between primary breast cancer diagnosis and LMD diagnosis for HR-positive breast cancers may also reflect a longer metastatic route that first includes a non-CNS location. In this study, we performed a retrospective analysis on 141 breast cancer patients with LMD treated at Stanford Hospital between 2008-2020. The Stanford LMD cohort was analyzed using linear regression analysis, Cox regression analysis, and Chi-squared analysis. HR status was recorded (when available) for local recurrences and distant metastases, and was included in our regression models as a time-dependent HR covariate. We tested if: (1) HR-negative breast cancers had shorter time between primary and LMD diagnosis as well as worse survival after LMD diagnosis versus HR-positive breast cancers, and if (2) HR loss (i.e., HR status changing from positive to negative) increased likelihood of the metastasis to CNS. In agreement with published studies, the Stanford LMD cohort showed that HR-negative cancers had shorter time to development of LMD (p = 0.0004 for ER, p = 0.0001 for PR) and increased mortality following LMD (p = 0.010 for ER, p = 0.009 for PR). We found that HR-negative status at primary breast cancer diagnosis increased risk of first metastasis to CNS versus non-CNS locations (p = 0.021 for ER, p = 0.043 for PR). HR status switch (also known as hormone discordance) between primary diagnosis and metastasis occurred in 15% of patients in Stanford cohort, consistent with other studies. Importantly, accounting for HR status switch strengthened the association between HR-negative status and the risk of CNS metastasis [Hazard Ratio = 2.506 (1.395 - 4.500) for “baseline ER status” versus Hazard Ratio = 3.509 (1.890 - 6.515) for “time-dependent ER status”, Hazard Ratio = 2.420 (1.310 - 4.471) for “baseline PR status” versus Hazard Ratio = 3.407 (1.815 - 6.395) for “time-dependent PR status”]. In conclusion, HR-negative status was associated with shorter time to development of LMD, higher CNS tropism, and shorter survival following LMD diagnosis. Taking into consideration hormone discordance between the primary tumor and metastatic sites strengthened this association. Our results suggest a potential value in testing HR status at all recurrences and advanced CNS monitoring following HR loss. Citation Format: Sophia Pribus, Maxine C. Umeh-Garcia, Bo Gu, Bryanna Godfrey, Summer Han, Sophia Chernikova, Melanie Hayden Gephart. Loss of hormone receptor expression in breast cancer is associated with increased brain tropism and accelerated progression of leptomeningeal disease [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 PO5-06-01.

Theranostic potential of a novel aptamer specifically targeting HER2 in breast cancer cells.

The overexpression of HER2 is correlated with poorer outcomes and therapeutic resistance in breast cancer patients. While HER2-targeted therapies have shown improvement, prognosis remains poor for HER2-positive breast cancer patients, and these treatments have limitations. Therefore, it is crucial to explore effective molecular strategies for early detection and treatment of HER2-positive breast cancers. In this study, we employed the cell-SELEX method to generate a selective aptamer capable of recognizing HER2 in its native conformation within breast cancer cells, for theranostic applications. Utilizing an adherent cell-SELEX approach, we developed and explored a DNA aptamer, named HMAP7, which can specifically target HER2 in the MDA-MB-453 and SK-BR-3 human breast cancer cell lines. After sequencing, the binding affinities of 10 candidate aptamers to HER2 receptors were evaluated by measuring fluorescence intensities within intact cells using near-infrared optical imaging. The dissociation constant of HMAP7 was determined to be in the nanomolar range in both cell lines. The cell-SELEX-derived aptamer sequence, HMAP7 (41-mer), exhibited the highest binding affinity and specificity for HER2. HMAP7 was rapidly internalized into breast cancer cells overexpressing HER2 but showed no uptake in the HER2 receptor-deficient breast cancer cell line MDA-MB-231. Moreover, HMAP7 demonstrated remarkable selectivity for HER2, rendering it suitable for use in complex biological systems. Our findings suggest that the novel DNA aptamer HMAP7 holds promise for both therapeutic and diagnostic applications, enabling selective delivery of therapeutic agents or imaging of HER2-positive breast tumors.

EFFECT OF COWANIN ON CYCLIN D1 EXPRESSION IN MCF-7/HER2 BREAST CANCER CELLS

Objective: Cowanin, isolated from the stem bark of Asam kandis (Garcinia cowa Roxb.) has been known to have cytotoxic activity in MCF-7/HER2 breast cancer cells. Recent studies have reported that cowanin compounds can inhibit MCF-7/HER2 cell migration and the T47D cell cycle in the G0-G1 phase. This research aims to determine the effect of cowanin on the cyclin D1 protein expression in MCF-7/HER2 breast cancer cells.
 Methods: The treatment consisted of a negative control group and a group given cowanin at a concentration of IC50 value (10,51 µM). The expression of cyclin D1 protein was detected using the western blot method. Observations of protein area and density were carried out using ImageJ software. Data were analyzed using the independent T-test.
 Results: The research showed that cowanin compounds induced cell cycle arrest of MCF-7/HER2 breast cancer cells by reducing the expression of cyclin D1 protein (p<0,05).
 Conclusion: The findings show that cowanin can significantly decrease the area and density of cyclin D1 protein

Evaluation of the influence of trastuzumab therapy on serum levels of HER-2 protein and breast cancer cell lines.

Breast cancer is a complex disease characterised by abnormal cell growth in breast tissue. Trastuzumab is a targeted therapy that inhibits the HER-2 receptor and suppresses tumour growth. We aimed to determine if the clinical course of the disease could be predicted by early changes in serum levels of human epidermal growth factor receptor 2 (HER-2/neu) following trastuzumab-based therapy. The study enrolled 120 women, divided into an experimental group (60 breast cancer patients receiving trastuzumab) and a control group (60 healthy women). Serum samples were collected before each weekly trastuzumab treatment. In addition, human breast cancer cell lines MCF-7 and AMJ13 were cultured in vitro and treated with trastuzumab. The study assessed cell viability using a cytotoxicity assay (methyl thiazolyl tetrazolium) and measured HER-2 protein levels. The half maximal inhibitory concentration (IC50) was determined to evaluate the effect of trastuzumab on breast cancer. The results showed that breast cancer patients had significantly lower serum levels of HER-2 compared to the control group. The cytotoxicity assay demonstrated that increasing trastuzumab concentration enhanced growth inhibition and cytotoxicity in the cell lines. There was a significant difference in IC50 between the MCF-7 and AMJ13 cell lines. The study provides valuable insights into the effects of trastuzumab on serum HER-2 levels and breast cancer cell lines. These findings have implications for resource allocation and treatment decisions in breast cancer management. By understanding the impact of trastuzumab on HER-2 levels and tumour cells, healthcare professionals can make more informed decisions regarding therapy options for patients.

MiR-18a-5p attenuates HER2-positive breast cancer development by regulating PI3K/AKT pathway

ABSTRACT Objective: Human epidermal growth factor receptor 2 positive (HER2+) breast cancer (BC) is associated with poor prognosis. This study aimed to elucidate the role of miR−18a−5p in regulation of HER2+-BC progression along with its mechanism of action. Methods: The expression of miR−18a−5p and HER2 in BC cells and tissues was analyzed using quantitative real-time PCR while protein level expression of AKT Serine/Threonine Kinase 1 (AKT), phosphorylated AKT (p-AKT), Phosphatidylinositol 3-kinase (PI3K), phosphorylated-PI3K (p-PI3K), and HER2 were assessed by western blotting. Cell Counting Kit−8, wound healing, and cell adhesion assays were used for in vitro analysis along with xenograft tumor model construction for in vivo analysis. Pearson correlation analysis and dual-luciferase reporter (DLR) assays were used to ascertain the targeting association between miR−18a−5p and HER2. Results: There was a downregulation of miR−18a−5p expression in the BC tissues and cells. Functionally, overexpression of miR−18a−5p prevented BC cells from proliferation, adherence, migration, and activation of the P-PI3K/P-AKT pathway. In vivo experiment revealed that tumor growth was suppressed when miR−18a−5p was overexpressed. In BC, HER2 overexpression increased cell proliferation, cell-cell adhesion, migration, and P-PI3K/P-AKT signaling, but overexpression of miR−18a−5p reversed this effect because of the target relationship between miR−18a−5p and HER2. Conclusion: miR−18a−5p inhibits HER2+ BC progression by targeting HER2 to inhibit PI3K/AKT pathway activation. A theoretical foundation for the identification of new therapeutic targets for HER2+ BC may be provided by the miR−18a−5p – HER2 axis.

Abstract P6-14-07: Genetic ablation of ACF7, a member of the +TIP family of microtubule-binding proteins, impairs metastasis of HER2-positive breast cancer

Abstract Breast cancer is the leading cause of cancer death in women worldwide. Among the different subtypes of this disease, HER2-positive breast cancer is known to be one of the most aggressive and is linked to poor prognosis. This subtype is prone to develop metastases, reflecting a significant cellular plasticity. While the molecular machinery controlling actin dynamics is well implicated in cell invasion, the contribution of the microtubules is ill-defined. We recently conducted a functional screen to identify such regulators, and characterized ACF7, a microtubule plus-end binding protein (+TIP), as a new promoter of invasion. Here, we aim to determine the in vivo contribution of ACF7 to tumor progression and to reveal the cellular and molecular mechanisms allowing this +TIP to promote metastasis. We analyzed the expression of ACF7 at the protein level across a panel of tumor microarrays and found that it is detectable in all breast cancer subtypes with the highest levels seen in HER2-positive. To study the role of ACF7 during tumor progression, we generated an ACF7 genetically engineered mouse model of breast cancer. We exploited a HER2-positive breast cancer mouse model (MMTV-NIC) that we bred with ACF7Flox mice. This approach allows for conditional deletion of ACF7 in mammary glands and represents a powerful model to define the roles of ACF7 in HER2-driven breast cancer. Our results show that ACF7 is not involved in tumor initiation and tumor growth as no difference between number of nodules per mouse, tumor mass or number of mammary intraepithelial neoplastic lesions was shown. However, our results show a significant decrease in lung metastasis in the ACF7cKO mice. To gain mechanistic insights into the roles of ACF7 in this specific process, we derived primary cell lines from tumors isolated from MMTV-NIC-ACF7WT and MMTV-NIC-ACF7cKO mice. We explored if ACF7 contributes to migration and invasion in a HER2 context. By using live-cell imaging, we demonstrated that ACF7-null cells display defects in both random and directed cell migration, as they show a delay in wound closure. We determined that ACF7KO cells show an increase in focal adhesions size, and exhibit defects in reorienting their actin and microtubules cytoskeletons parallel to the direction of migration. ACF7 was also found to promote cell invasion since ACF7KO cells were less efficient to cross a Matrigel membrane. To investigate the metastatic potential of ACF7-null tumors, we performed RNA-sequencing using RNA isolated from MMTV-NIC-ACF7WT and MMTV-NIC-ACF7cKO tumors. We identified a striking change in expression of genes involved in epithelial to mesenchymal transition (EMT), such as for example E-cadherin, Vimentin or the transcription factor Twist. These results suggest that ACF7 promotes metastasis through maintaining an EMT state in HER2 breast cancer cells. Collectively, our work demonstrates ACF7 as an important player in HER2 positive breast cancer progression via its functions in both cell migration and cell invasion. This works also suggests that developing approaches to therapeutically target regulators of microtubule dynamics may reveal new opportunities to decrease tumor progression and metastatic expansion. Citation Format: Rebecca Cusseddu, Jean-François Côté. Genetic ablation of ACF7, a member of the +TIP family of microtubule-binding proteins, impairs metastasis of HER2-positive breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-14-07.

3D‐Cell Spotter Cooperated High‐Throughput Screening (HTS) for Estimating Targeted Drugs in Breast Cancer Cells

AbstractA 3D‐cell spotter is developed to maximize the efficiency of 3D cell‐based high‐throughput screening (3D‐HTS). The performance of 3D‐HTS is verified through heatmap analysis for distinguishing responses to targeted drugs according to the expression of HER2 in breast cancer cells. Under 2D culture, the expression of cell proliferation receptors is lost as cells adhere to the bottom of the plate. To address this issue, alginate is applied as an extracellular matrix to the 3D cell culture to maintain the expression of tumor‐associated proteins. In general, higher drug resistance is observed with 3D‐HTS. However, increased drug resistance does not necessarily mean decreased analytical sensitivity to drug responsiveness. In particular, with 3D‐HTS, EGFR‐targeted drug reactivity to HER2‐positive and ‐negative cell lines are distinguished by a higher positive/negative (P/N) score, and more combinations of strong correlations between EGFR‐targeted drugs are observed. After confirming the reactivity of 65 drugs using previous analysis results with heatmap analysis, two new non‐EGFR‐targeted drugs with excellent efficacy against breast cancer cells are identified. To verify this, high‐content imaging analysis is conducted to assess their ability to inhibit phospho‐EGFR activity. Therefore, the 3D‐HTS‐based heatmap analysis method is useful for analyzing the efficacy of targeted drugs against breast cancer cells.

IKZF3 amplification frequently occurs in HER2-positive breast cancer and is a potential therapeutic target.

Breast cancer is one of the leading causes of cancer death in women, and although treatment outcome has substantially improved in the past decades, advanced or metastatic breast cancers still carry a poor prognosis. Gene amplification is one of the frequent genetic alterations in cancer, and oncogene amplification may be associated with cancer aggressiveness and oncogenicity. Targeting amplified genes such as HER2 has vastly improved disease outcome and survival, and anti-HER2 therapeutics have revolutionized the standard of care in HER2 breast cancer. Besides currently known druggable gene amplifications including ERBB2 and FGFR2, other frequently amplified genes are relatively less well known for function and clinical significance. By querying four large databases from TCGA and AACR-Genie, from a total of 11,890 patients with invasive ductal breast carcinoma, we discover IKZF3, CCND1, ERBB2 to be consistently amplified across different cohorts. We further identify IKZF3 as a frequently amplified gene in breast cancer with a prevalence of 12-15% amplification rate. Interestingly, IKZF3 amplification is frequently co-amplified with ERBB2/HER2, and is also associated with worse prognosis compared to IKZF3 non-amplified cancers. Analysis of HER2 breast cancer patients treated with trastuzumab revealed decrease in both ERBB2/HER2 and IKZF3 expression. Further investigation using the DepMap for gene dependency by genome-wide CRISPR screening revealed dependence on IKZF3 in HER2 breast cancer cell lines. Our study utilized an integrative analysis of large-scale patient genomics, transcriptomics and clinical data to reveal IKZF3 as a frequently amplified gene, and suggest a potential role of IKZF3 as a druggable target for HER2 breast cancer.

Anti‐proliferative effects of pentagamaboronon‐0‐sorbitol on HER2‐overexpressing breast cancer cells

HER2‐positive breast cancer is an aggressive form of the disease that is associated with poor prognosis and chemo‐resistance. As such, investigation continues into the development of a new HER2‐targeted drug for breast cancer. This study investigated the anti‐proliferative activities of pentagamaboronon‐0‐sorbitol (PGB‐0‐So) in HER2‐overexpressing breast cancer (MCF‐7/HER2) cells. The cytotoxicity of PGB‐0‐So was assessed via MTT assay. Flow cytometry with propidium iodide and annexin‐V‐FITC staining was conducted to investigate the mechanism of PGB‐0‐So in inhibiting the proliferation of MCF‐7/HER2 cells. Finally, FACS analysis with 2′,7′–dichlorofluorescin diacetate staining was performed to examine intracellular ROS production. PGB‐0‐So exerted cytotoxicity towards MCF‐7/HER2 breast cancer cells with an IC50 value of 36 μM. PGB‐0‐So induced S‐phase arrest and apoptosis in MCF‐7/HER2 cells. Moreover, PGB‐0‐So could increase intracellular ROS production in MCF‐7/HER2 cells. PGB‐0‐So exerted anti‐proliferative activity towards MCF‐7/HER2 cells. This compound may be developed as a chemotherapeutic agent against HER2‐overexpressing breast cancer.

Gasdermin B over-expression modulates HER2-targeted therapy resistance through LC3B/Rab7 interaction

Tumor resistance to therapy is one of the most important challenges in current oncology. A clear example of this major issue is the HER2-positive tumors characterized by ERBB2 (HER2/neu) overexpression. HER2+ overexpression/ amplification occurs in several tumors, especially in breast and gastric carcinomas that show an aggressive phenotype traditionally associated to poor prognosis. In fact, despite the clinical outcome of these tumors has gradually improved, due to the development of different antiHER2 therapies, many patients will not respond to these treatments or develop acquired resistance. Several potential resistance mechanisms to these targeted therapies have been described. Among them, our laboratory established, for the first time, that the amplification/overexpression of Gasdermin B (GSDMB), which occurs in more than 60% HER2+ breast cancer patients, is a marker of poor prognosis. Moreover, our data in HER2+ tumors revealed that GSDMB overexpression correlated to distant metastasis, poor clinical outcome, and reduced response to anti-HER2 therapies. In this context, we have attempted to elucidate the molecular mechanism whereby GSDMB collaborates in the resistance to anti-HER2 therapies. To decipher the functional relevance of GSDMB in promoting resistance to HER2-targeted therapies we performed several molecular approaches (immunoblot, flow cytometry, immunoprecipitation, etc.) in different breast and gastric carcinoma cell models. Finally, we validated the efficacy of the identified targeted treatment using two complementary in vivo preclinical models (mice and zebrafish). Our results confirmed that similarly to HER2 breast tumors, GSDMB is also over-expressing in more than 50% in HER2 gastric tumors. Furthermore, this over-expression renders HER2 breast and gastric cancer cells more resistant to anti- HER2 agents by promoting protective autophagy. Consistent with this, we proved that the combination of lapatinib with the autophagy inhibitor chloroquine increases the therapeutic response specifically in GSDMB-positive tumors in vitro and in vivo. Mechanistically, we also confirmed that GSDMB forms a complex with LC3B and Rab7. Finally, we validated these results in clinical samples of HER2 breast and gastric cancers, where GSDMB/LC3B/Rab7 co- expression associates significantly with relapse. In conclusion, our data decipher the molecular mechanism that underlies the relation between Gasdermin B and pro-survival autophagy with the final goal of finding a therapy that would be effective in overcoming resistance to anti-HER2 standard therapies in HER2/GSDMB+ tumors.

The Precise Detection of HER-2 Expression in Breast Cancer Cell via Au25 Probes.

Triple-negative breast cancer (TNBC) accounts for nearly one-quarter of all breast cancer cases, but effective targeted therapies for this disease remain elusive because TNBC cells lack the expression of the most common three receptors seen in other subtypes of breast cancers. The medium-term diagnosis of breast cancers is essential for development and prognosis. According to reports, patients with TNBC may be converted to a positive epidermal growth factor receptor 2(HER-2) after chemotherapy, and trastuzumab treatment will have a better prognosis. Therefore, it is important to accurately quantify the expression of HER-2 in breast cancer cells. Herein, we design a red fluorescent Au25 probe synthesized with BSA-biotin as the ligand, which is accurately quantified by HER-2 primary antibody-biotin using the avidin system. The quantitative detection of the expression of HER-2 in breast cancers is helpful for the companion diagnostic of breast cancer treatment and provides follow-up treatment.

Inhibiting ERK5 overcomes breast cancer resistance to anti-HER2 therapy by targeting the G1/S cell cycle transition.

Targeting the human epidermal growth factor receptor 2 (HER2) became a landmark in the treatment of HER2-driven breast cancer. Nonetheless, the clinical efficacy of anti-HER2 therapies can be short-lived and a significant proportion of patients ultimately develop metastatic disease and die. One striking consequence of oncogenic activation of HER2 in breast cancer cells is the constitutive activation of the extracellular-regulated protein kinase 5 (ERK5) through its hyperphosphorylation. In this study, we sought to decipher the significance of this unique molecular signature in promoting therapeutic resistance to anti-HER2 agents. We found that a small-molecule inhibitor of ERK5 suppressed the phosphorylation of the retinoblastoma protein (RB) in HER2 positive breast cancer cells. As a result, ERK5 inhibition enhanced the anti-proliferative activity of single-agent anti-HER2 therapy in resistant breast cancer cell lines by causing a G1 cell cycle arrest. Moreover, ERK5 knockdown restored the anti-tumor activity of the anti-HER2 agent lapatinib in human breast cancer xenografts. Taken together, these findings support the therapeutic potential of ERK5 inhibitors to improve the clinical benefit that patients receive from targeted HER2 therapies.

Abstract P5-08-09: Characterization of the Rac/Cdc42 inhibitor MBQ-168 as an anti-cancer compound

Abstract Metastasis continues to be the primary cause of cancer-related death in women with breast cancer, needing targeted therapies to inhibit this process. Therefore, we developed inhibitors targeting the homologous Rho GTPases Rac1 and Cdc42, which direct the actin cytoskeletal changes required for cell migration/invasion; and thus, metastasis. We characterized the potent inhibitor, MBQ-167 that blocks both Rac1 and Cdc42 at IC50s 103 nM and 78 nM, respectively, in human HER2 (+) and triple-negative breast cancer (TNBC) cells. Consequently, MBQ-167 inhibits tumor growth and metastasis by ~90% in pre-clinical mouse models of breast cancer (Humphries-Bickley, et al., 2017). To improve the activity and solubility of MBQ-167, we synthesized a panel of derivatives, maintaining the 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole as the core for each derivative. Of the derivatives tested, MBQ-168 demonstrated improved solubility and comparable efficacy. Similar to MBQ-167, MBQ-168 induced actin cytoskeletal disintegration, cell rounding, and detachment to ultimately undergo anoikis. In 120hrs, MBQ-168 significantly inhibited the viability of MDA-MB-231 at a GI50 of 228nM and HER2+ MDA-MB-435 cells with a GI50 of 137 nM, without affecting the viability of Human Mammary Epithelial Cells (HMEC). In apoptosis assays, MBQ-168 treatment, at 500nM for 48 hrs, demonstrated a similar response to MBQ-167 by >4-fold increase in Caspase 3/7 activity in HER2 (+), and MDA-MB-231 and MDA-MB-468 TNBC cells. In wound healing assays, MBQ-168 also responded similar to MBQ-167 by a ~80% inhibition of breast cancer cell migration at 250nM and 500nM for 24hrs. Moreover, as quantified from Rac1.GTP pulldown assays, following 250nM treatment for 24hrs, MBQ-168 inhibited Rac1 activation in the attached cell population by ~70%, and in the detached cell population by ~100%, demonstrating a potent inhibition of Rac1 activation. In a preliminary study for relative efficacy in mice, MBQ-168 significantly reduced HER2+ MDA-MB-435 mammary tumor growth (~90%) at 5mg/kg BW via IP administration. In conclusion, MBQ-168 is an effective Rac inhibitor that reduces breast cancer cell viability, induces apoptosis, and inhibits breast cancer cell migration, actin cytoskeletal extensions, and Rac1 activation to result in a drastic reduction in mammary tumor growth in mice. We predict that the increased solubility of MBQ-168 will make it more bioavailable than MBQ-167. Thus, we have demonstrated that small structural modifications of MBQ-167 can affect the cytotoxic activity of carbazole derivatives with potential as anti-cancer drugs, and as tools to block Rac/Cdc42 activities in biological systems. Citation Format: Julia Medina, Tatiana Matos, Luis Velazquez, Michael Rivera, Ailed Cruz-Collazo, Eliud Hernandez, Cornelis Vlaar, Suranganie Dharmawardhane. Characterization of the Rac/Cdc42 inhibitor MBQ-168 as an anti-cancer compound [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-09.

Delivery Capacity and Anticancer Ability of the Berberine-Loaded Gold Nanoparticles to Promote the Apoptosis Effect in Breast Cancer.

Simple SummaryIn this research, we aimed to evaluate the biological effects of physically gold nanoparticle-collagen nanocarrier incorporated with alkaloid berberine (Au-Col-BB) on non-transformed bovine aortic endothelial cells (BAEC) and Her-2 breast cancer cell lines through in vitro and in vivo assessments. Au-Col-BB showed better cytotoxicity, as well as significantly induced cell apoptosis in Her-2 cancer cells compared with normal cells (non-transformed BAEC). Further, Au-Col-BB also demonstrated better anti-cancer capacity for inhibiting cell growth in Her-2 tumor-bearing mice. In brief, we confirmed that the Au-Col-BB nanocarrier could be a potential nanodrug for increasing the efficiency of specific therapeutic effects in breast cancer disease.Gold nanoparticles (AuNPs) were fabricated with biocompatible collagen (Col) and then conjugated with berberine (BB), denoted as Au-Col-BB, to investigate the endocytic mechanisms in Her-2 breast cancer cell line and in bovine aortic endothelial cells (BAEC). Owing to the superior biocompatibility, tunable physicochemical properties, and potential functionalization with biomolecules, AuNPs have been well studied as carriers of biomolecules for diseases and cancer therapeutics. Composites of AuNPs with biopolymer, such as fibronectin or Col, have been revealed to increase cell proliferation, migration, and differentiation. BB is a natural compound with impressive health benefits, such as lowering blood sugar and reducing weight. In addition, BB can inhibit cell proliferation by modulating cell cycle progress and autophagy, and induce cell apoptosis in vivo and in vitro. In the current research, BB was conjugated on the Col-AuNP composite (“Au-Col”). The UV-Visible spectroscopy and infrared spectroscopy confirmed the conjugation of BB on Au-Col. The particle size of the Au-Col-BB conjugate was about 227 nm, determined by dynamic light scattering. Furthermore, Au-Col-BB was less cytotoxic to BAEC vs. Her-2 cell line in terms of MTT assay and cell cycle behavior. Au-Col-BB, compared to Au-Col, showed greater cell uptake capacity and potential cellular transportation by BAEC and Her-2 using the fluorescence-conjugated Au-Col-BB. In addition, the clathrin-mediated endocytosis and cell autophagy seemed to be the favorite endocytic mechanism for the internalization of Au-Col-BB by BAEC and Her-2. Au-Col-BB significantly inhibited cell migration in Her-2, but not in BAEC. Moreover, apoptotic cascade proteins, such as Bax and p21, were expressed in Her-2 after the treatment of Au-Col-BB. The tumor suppression was examined in a model of xenograft mice treated with Au-Col-BB nanovehicles. Results demonstrated that the tumor weight was remarkably reduced by the treatment of Au-Col-BB. Altogether, the promising findings of Au-Col-BB nanocarrier on Her-2 breast cancer cell line suggest that Au-Col-BB may be a good candidate of anticancer drug for the treatment of human breast cancer.

Characterization of Rac/Cdc42 Inhibitors as Anti‐cancer Compounds

Metastatic breast cancer continues to be the primary cause of cancer-related death in women, needing targeted therapies to inhibit this process. Therefore, we are designing small molecule inhibitors to specifically block the metastatic phase, focusing on the Rho GTPases Rac1 and Cdc42. These close homologs direct the actin cytoskeletal changes required for cell migration/invasion, driving the first steps of metastasis. Rho GTPases are activated by Guanine Nucleotide Exchange Factors (GEF), which promote the exchange of GDP to GTP. We have designed the molecule Ehop-016, a carbazole derivative, to inhibit Rac1 at an IC50 of 1.1 uM by disrupting the interaction of the GEF Vav2 with Rac1. Based on this molecule, we designed a more potent inhibitor, MBQ-167, to block both Rac1 and Cdc42 at IC50s 103 nM and 78 nM, respectively,in human HER2 (+) breast cancer cells and triple-negative breast cancer cells. Moreover, MBQ-167 inhibits tumor growth and metastasis by ~90% in pre-clinical mouse models of breast cancer. MBQ-167 also has a different mechanism of inhibition to EHop-016 by direct inhibition of GTP/GDP binding. We hypothesized that by designing stronger inhibitors based on MBQ-167 structure with drug-like properties, we could achieve an improved inhibition for Rac1 and Cdc42, as well as achieve different specificities against the numerous oncogenic GEFs that regulate cancer malignancy. Thus, we synthesized a panel of derivatives, maintaining 9-ethyl-3-(1H-1,2,3-triazol-1-yl)-9H-carbazole as the core for each derivative. HER2 (+) breast cancer cells were treated with 500 nM of the derivatives for 72 hrs. The CellTiter 96 Non-Radioactive Cell Proliferation Assay was used to analyze cell viability by quantifying both attached and detached cells in the same well. Results show that MBQ-167 derivatives decreased cell viability to various degrees. Therefore, we have demonstrated that small structural modifications of MBQ-167 can affect the cytotoxic activity of carbazole derivatives with potential as anti-cancer drugs, and as tools to block Rac/Cdc42 activities in biological systems.

The Effect of Sirtuin 2 (Sirt2) Overexpressing Bone Marrow Mesenchymal Stem Cells on the Growth of Human Epidermal Growth Factor Receptor 2 (Her-2) Breast Cancer Cells and Its Mechanism

Our study investigates the effect of high expression of Sirt2 in MSCs (MSCs-Sirt2) on Her-2 breast cancer cell proliferation. A mouse subcutaneous xenograft tumor model was established and MSCssirt2 analysis was performed on nude mice. TUNEL staining, flow cytometry, western-blot, real-time PCR and immunohistochemistry were used to detect cancer cell apoptosis. The number of NK cells infiltrated by flow cytometry detected the tumor tissue of tumor-bearing mice, and its killing activity on tumor-bearing mice was detected by isotope labeling and release method. The levels of TNF-α, IFN-γ, IL-8, IL-6 and IL-10 were detected by ELISA. Caspase-3 level was decreased in the MSCs group (P <0.01) while increased in the MSCs-sirt2 group (P <0.001). However, PCNA expression showed an opposite profile in the Her-2 group and MSCs-sirt2 group compared to Caspase-3 level (P <0.01). The tumor volume and weight in the MSCs-sirt2 group was significantly reduced (P < 0.01), while increased in the MSCs group significantly (P < 0.05). The number of Ki-67-positive tumor cells in MSCs-sirt2 group was significantly reduced (P <0.01) and increased in MSCs group (P < 0.001) with oppositive number of TUNEL-positive tumor cells in the MSCs-sirt2 group and MSCs group (P <0.01). IFN-γ level showed an upward trend (P <0.001). The NK cell toxicity of MSCs-Sirt2 group was significantly higher (P <0.001). MSCs-Sirt2 has an inhibitory effect on Her-2 breast cancer cell growth by enhancing the local inflammatory response of NK cells.

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer

BackgroundAround 15–20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance.MethodsRANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling.ResultsRANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status.ConclusionsOur data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated.

Abstract PO-080: Deep learning-based analysis of heterogeneity of breast cancer cells using lens-free digital in-line holography

Abstract Lens-free digital in-line holography (LDIH) produces cellular diffraction patterns from a large field of view that lens-based microscopes cannot achieve. It is a promising diagnostic tool allowing comprehensive cellular analysis with high-throughput capability. Since these diffraction images are far more complicated to discern, conventionally computational algorithms are used to reconstruct cellular images for the diffraction patterns. However, it is inefficient and prone to errors. Here, we developed a deep learning architecture, HoloNet to directly analyze the diffraction patterns from LDIH. In addition to the standard CNN (Convolutional Neural Network), the HoloNet includes a holo-branch that extracts large features from holograms and integrate them with the small features from the standard CNN. Using HoloNet, we accurately predicted the intensity values of ER/PR and HER2 in breast cancer cells and classified four known subtypes of breast cancer cells. Moreover, we applied a HoloNet dual embedding, which learns high-level diffraction features related to breast cancer cell types and the intensities of ER/PR and HER2. This allowed us to identify potential subtypes of breast cancer cells from the cell lines and the fine needle aspiration samples from breast cancer patients. We demonstrate that our HoloNet can enable LDIH to perform more detailed and rapid analyses of subtypes of breast cancer cells. Citation Format: Tzu-Hsi Song, Mengzhi Cao, Jouha Min, Hyungsoon Im, Hakho Lee, Kwonmoo Lee. Deep learning-based analysis of heterogeneity of breast cancer cells using lens-free digital in-line holography [abstract]. In: Proceedings of the AACR Virtual Special Conference on Artificial Intelligence, Diagnosis, and Imaging; 2021 Jan 13-14. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(5_Suppl):Abstract nr PO-080.

Determining the Efficiency of Single Molecule Quantum Dot Labeling of HER2 in Breast Cancer Cells.

Quantum dots exhibit unique properties compared to other fluorophores, such as bright fluorescence and lack of photobleaching, resulting in their widespread utilization as fluorescent protein labels in the life sciences. However, their application is restricted to relative quantifications due to lacking knowledge about the labeling efficiency. We here present a strategy for determining the labeling efficiency of quantum dot labeling of HER2 in overexpressing breast cancer cells. Correlative light- and liquid-phase electron microscopy of whole cells was used to convert fluorescence intensities into the underlying molecular densities of the quantum dots. The labeling procedure with small affinity proteins was optimized yielding a maximal labeling efficiency of 83%, which was applicable to the high amount of ∼1.5 × 106 HER2 per cell. With the labeling efficiency known, it is now possible to derive the absolute protein expression levels in the plasma membrane and its variation within a cell and between cells.