Hormone-dependent Breast Cancer Research Articles

Current treatment aspects of hormone-dependent ERBB2-negative metastatic breast cancer: overall survival outcomes

This review provides an overall survival analysis in patients with hormone-dependent ERBB2-negative metastatic breast cancer (mBC) treated with CDK4/6 inhibitors combined with endocrine therapy (ET). The results of MONALEESA-2, 3, 7, PALOMA-2, 3, and MONARCH-2, 3 studies are provided.
 The emergence of a new class of drugs, CDK4/6 inhibitors, opened the way to revise the treatment algorithm for patients with HR+/HER2- mBC. Currently, in treatment lines 1 and 2, the combination of CDK4/6 inhibitors with ET is preferable. The proven overall survival advantage in treatment lines 1 and 2, and data from an exploratory analysis subgroup, demonstrate the efficacy of ribociclib in combination with ET. The results of the studies confirm the benefits of combination therapy in routine practice to treat HR+/HER2- mBC regardless of the patient's age, menopausal status, ET variant, and disease prevalence.

Computational and In Vitro Approaches to Elucidate the Anti-cancer Effects of Arnica montana in Hormone-Dependent Breast Cancer.

Breast cancer is the most common cancer in women worldwide. Use of homeopathic medicines for the treatment of cancers has increased in the last several years. Arnica montana is an anti-inflammatory homeopathic medicine used in traumatic conditions and because of this property we performed investigations for its potential as a chemotherapeutic agent against breast cancer. An ethanolic extract of Arnica montana (mother tincture, MT), prepared according to the Homoeopathic Pharmacopoeia of India, was characterized by gas chromatography-mass spectroscopy (GC-MS), followed by computational (in silico) analysis using molecular docking, to identify specific compounds that can bind and modulate the activity of key proteins involved in breast cancer survival and progression. To validate the in silico findings, in a controlled experiment breast cancer cells (MCF7) were treated in vitro with Arnica montana and the cytotoxic effects assessed by flowcytometry, fluorescence microscopy, scratch assay, clonogenic potential and gene expression analysis. Phytochemical characterization of ethanolic extract of Arn MT by GC-MS allowed identification of several compounds. Caryophyllene oxide and 7-hydroxycadalene were selected for molecular docking studies, based on their potential drug-like properties. These compounds displayed selective binding affinity to some of the recognized target proteins of breast cancer, which included estrogen receptor alpha (ERα), progesterone receptor (PR), epidermal growth factor receptor (EGFR), mTOR (mechanistic target of rapamycin) and E-cadherin. In vitro studies revealed induction of apoptosis in MCF7 cells following treatment with Arn MT. Furthermore, treatment with Arn MT revealed its ability to inhibit migration and colony forming abilities of the cancer cells. Considering the apoptotic and anti-migratory effects of Arnica montana in breast cancer cells in vitro, there is a need for this medicine to be further validated in an in vivo model.

Evolution of the CDK4/6 inhibitor abemaciclib: from palliative to adjuvant therapy. Clinical experience with abemaciclib in patients with hormone-receptor-positive, HER2-negative breast cancer

Introduction. Cyclin-dependent kinase 4/6 inhibitors are indicated in endocrine therapy for the treatment of hormone receptorpositive, HER2-negative, advanced, or metastatic breast cancer. In the recent past, abemaciclib made its debut as a combinatorial partner for adjuvant therapy in hormone-dependent breast cancer. This article demonstrates the analysis of our own experience of introducing abemaciclib into clinical practice.Aim. The aim of the study was to evaluate the preliminary results of treatment of patients with hormone receptor-positive HER2-negative metastatic breast cancer using abemaciclib outside the framework of clinical trials, in the real clinical practice of an oncological dispensary.Materials and methods. A retrospective analysis of the results of treatment was carried out in 27 patients who were prescribed abe-maciclib in various regimens and lines from April 2021 to January 2022 in the conditions of routine practice in the Krasnodar region.Results and discussion. Analysis of the total population of 27 patients at a median follow-up of 8 months showed that the median PFS was not reached. The one-year PFS was 68.8%. Treatment outcomes were independent of prescribing abemaciclib to treat baseline metastatic disease or breast cancer progression after previous radical treatment (p = 0.60). The PFS did not depend on the expression of progesterone receptors in the tumor tissue (p = 0.326) and the proliferative activity index Ki-67 (p = 0.618). Patients who received no more than 2 lines of previous drug treatment for a history of metastatic breast cancer had the greatest benefit from abemaciclib therapy (p = 0.001).Conclusions. Despite a relatively difficult group of patients (96% of patients with visceral metastases), abemaciclib has demonstrated efficacy and safety. The effectiveness did not depend on the analyzed factors: Ki-67, the level of expression of progesterone receptors, the type of metastatic disease (de novo metastatic or progressive breast cancer). In view of the best results, it is advisable to prescribe abemaciclib in the early lines of therapy.

PSPC1 is a potential prognostic marker for hormone-dependent breast cancer patients and modulates RNA processing of ESR1 and SCFD2

Breast cancer is the most common cancer type among women worldwide. The majority of breast cancer expresses estrogen receptor (ER) and endocrine therapy is a standard treatment of ER-positive breast cancer. However, development of the therapy resistance is still a major challenge and thus new therapeutic approaches are needed. Here we show that an RNA-binding protein, PSPC1, play a crucial role in ER-positive breast cancer growth through post-transcriptional gene regulation. We showed that siRNA-mediated PSPC1 silencing suppressed the proliferation of ER-positive breast cancer cells. Strong immunoreactivity (IR) of PSPC1 was correlated with poor prognosis for ER-positive breast cancer patients. Using immunoprecipitation, RNA-immunoprecipitation (RIP) and quantitative PCR (qPCR) experiments, we showed that PSPC1 interacted with PSF and was involved in post-transcriptional regulation of PSF target genes, ESR1 and SCFD2. Strong SCFD2 IR was correlated with poor prognosis for ER-positive breast cancer patients and combinations of PSPC1, PSF, and SCFD2 IRs were potent prognostic factors. Moreover, we identified DDIAS and MYBL1 as SCFD2 downstream target genes using microarray analysis, and finally showed that SCFD2 silencing suppressed tamoxifen-resistant breast tumor growth in vivo. These results indicated that PSPC1 and SCFD2 axis could be a promising target in the clinical management of the disease.

SARS-CoV-2 M Protein Facilitates Malignant Transformation of Breast Cancer Cells.

Coronavirus disease 2019 (COVID-19) has spread faster due to the emergence of SARS-CoV-2 variants, which carry an increased risk of infecting patients with comorbidities, such as breast cancer. However, there are still few reports on the effects of SARS-CoV-2 infection on the progression of breast cancer, as well as the factors and mechanisms involved. In the present study, we investigated the impact of SARS-CoV-2 proteins on breast cancer cells (BCC). The results suggested that SARS-CoV-2 M protein induced the mobility, proliferation, stemness and in vivo metastasis of a triple-negative breast cancer (TNBC) cell line, MDA-MB-231, which are involved in the upregulation of NFκB and STAT3 pathways. In addition, compared to MDA-MB-231 cells, the hormone-dependent breast cancer cell line MCF-7 showed a less response to M protein, with the protein showing no effects of promoting proliferation, stemness, and in vivo metastasis. Of note, coculture with M protein-treated MDA-MB-231 cells significantly induced the migration, proliferation, and stemness of MCF-7 cells, which are involved in the upregulation of genes related to EMT and inflammatory cytokines. Therefore, SARS-CoV-2 infection might promote the ability of aggressive BCC to induce the malignant phenotypes of the other non-aggressive BCC. Taken together, these findings suggested an increased risk of poor outcomes in TNBC patients with a history of SARS-CoV-2 infection, which required a long-term follow-up. In addition, the inhibition of NFκB and STAT3 signaling pathways is considered as a promising candidate for the treatment of worsen clinical outcomes in TNBC patients with COVID-19.

Aromatase Inhibitors and Risk of Metabolic and Cardiovascular Adverse Effects in Breast Cancer Patients-A Systematic Review and Meta-Analysis.

Aromatase inhibitors (AIs) have been considered first-line therapy for patients with hormone-dependent breast cancer due to their high efficacy and good tolerability. However, AIs are not free of adverse events, and studies show that therapy with AIs is associated with an increased risk of cardiovascular events and the development of insulin resistance and diabetes. We searched the Cochrane Central Register of Controlled Trials, PubMed and EMBASE up to 27 October 2020 for the prevalence of cardiovascular and/or metabolic adverse effects during treatment with AIs in postmenopausal women with breast cancer. A meta-analysis was performed using a random effects model. Odds ratios and 95% confidence intervals were calculated and illustrated using forest plot charts. We performed separate analyses depending on trial design. Twenty two studies met the inclusion criteria. AIs were associated with a higher risk of cardiovascular events, especially when we compared study arms in which AIs were used (alone or in sequence with TAM) with the arms in which TAM was used alone (OR = 1.16; 95%CI 1.04–1.30) or when comparing patients taking AIs alone to patients taking TAM alone or in sequence with AIs (OR = 1.24; 95%CI 1.11–1.38). A pooled analysis of five trials comparing adjuvant AIs to TAM showed the odds for arterial hypertension being 1.31 times higher for patients taking AIs; however, this did not reach statistical significance (OR = 1.31; 95%CI 0.47–3.65). We have not shown an increased risk of dyslipidemia or weight gain with the use of AIs. Our results suggest that postmenopausal women with breast cancer treated with AIs have an increased risk of cardiovascular events in comparison with TAM, potentially due more to a cardioprotective effect of the latter than the cardiotoxicity of AIs. We were unable to prove a similar association for hypertension, dyslipidemia, hyperglycemia or weight gain. Further high-quality RCTs and post-marketing safety observational studies are needed to definitively evaluate the impact of AIs on metabolic disorders in breast cancer patients.

Human epidermal growth factor receptor-2 and endocrine resistance in hormone-dependent breast cancer.

Endocrine therapies are the main treatment strategies for the clinical management of hormone-dependent breast cancer. Despite prolonged time to recurrence in the adjuvant setting and the initial clinical responses in the metastatic setting, many patients eventually encounter tumour relapse due to acquired resistance to these agents. Other patients experience a lack of tumour regression at the beginning of treatment indicating de novo resistance that significantly limits its efficacy in the clinic. There is compelling evidence that human epidermal growth factor receptor-2 (HER2) overexpression contributes to resistance to endocrine therapies in oestrogen receptor-positive (ER+) breast cancer. ER+/HER2+ tumours comprise about 10% of all breast cancer cases and about 60% of the whole set of HER2+ tumours. Most patients with primary ER+/HER2+ disease will receive antibody-based HER2-targeted therapy, but this is generally for no more than one year while endocrine treatment is usually for at least 5 years. A number of HER2-kinase inhibitors are also now in clinical use or in clinical trials, and the interaction of these with endocrine treatment may differ from that of antibody treatment. In this review article, we aim to summarise knowledge on molecular mechanisms of breast cancer resistance to endocrine therapies attributable to the impact of HER2 signalling on endocrine sensitivity, to discuss data from clinical trials addressing the role of HER2 in the development of endocrine resistance in the metastatic, neoadjuvant and adjuvant settings and to explore rational new therapeutic strategies.

Design and Synthesis of Bi‐aryl Methylated Lactam Derivatives to Inhibit the BRD7 Bromodomain Function in Prostate Cancer

Targeting bromodomain‐containing proteins has emerged as a promising therapeutic strategy and multiple clinical studies indicate that bromodomain (BD) inhibitors can act as anticancer and anti‐inflammatory agents. Bromodomain‐containing protein 7 (BRD7) is a subunit of the SWI/SNF chromatin remodeling complex, which is involved in the fine‐tuning of gene expression through changes in the organization of DNA. It regulates the progression of different types of cancers such as nasopharyngeal carcinoma and hormone‐dependent breast and ovarian cancers. BRD7 is also implicated in progression of prostate cancer, another hormone‐dependent malignancy; however, the role of BRD7 in this type of cancer is undefined. (Figure 1) We have characterized BRD7 as an oncogenic driver across a variety of prostate cancer cell lines, implicating BRD7 inhibitors as a potential therapeutic strategy to combat therapy resistant cancers.To develop inhibitors of BRD7 we are developing small molecules that bind selectively to the bromodomain of BRD7. The main challenge when developing BRD7 BD inhibitors is the high similarity to the BRD9 BD, its closest homolog. Several dual inhibitors of BRD7 and BRD9 have been developed, all of them being more selective for the BRD9 BD over the BRD7 BD. Using the X‐ray crystal structure of both BDs bound to a BRD9‐selective inhibitor, we identified an accessible hydrophobic region in the binding pocket of BRD7 that is not present in BRD9, which we hypothesized could be exploited to develop BRD7‐selective inhibitors. Based on this structural difference in the BRD7 binding pocket, we designed a library of compounds that share a bi‐aryl methylated lactam core. (Figure 2)From in silicomolecular docking simulations and in vitro affinity screenings, we have identified several ligands selective for BRD7. These compounds also display potent anti‐proliferative effects in prostate cancer cell lines, providing further evidence for proposing BRD7 as a therapeutic target in prostate cancer. Our on‐going work focuses on optimizing the potency and solubility properties of BRD7 inhibitors, and characterizing their selectivity using cell‐based models.

Triazoles Synthesis & Applications as Nonsteroidal Aromatase Inhibitors for Hormone-Dependent Breast Cancer Treatment

In the last few years, nonsteroidal aromatase inhibitors (AIs) have been emerged as promising agents for treating hormone-dependent breast cancer in postmenopausal women because of their inhibitory effect on estrogen synthesis. Indeed, these compounds can block the activity of aromatase, the enzyme that intervenes in the last steps of estrogen production pathway. Triazoles are the core structures of nonsteroidal AIs. The nitrogen atom of the triazole moiety plays a fundamental role in the aromatase functionality by interacting with the iron ions of the heme group. In general, AIs possess numerous advantages as they quench the last step of estrogen synthesis without any inhibitory effects on the production of other steroids produced via the same pathway. Some AIs as anastrozole, letrozole, and vorozole have already been approved by the Food and Drug Administration in the treatment of breast cancer. The previously mentioned compounds present severe and adverse effects as polycystic ovary syndrome (PCOS), resistance onset on long-term treatments, and a higher risk of bone fractures. This review focuses intensively on the role of AIs in the treatment of hormone-sensitive types of cancers, especially the role of triazoles as nonsteroidal AIs. Also, the review provides an overview about the chemistry of triazoles along with the different methods by which the v -triazoles and s-triazoles are synthesized.

Modulatory potential of Tamarindus indica seed coat on oestrogen and progestone secretion in MCF-7 cell lines

According to epidemiological research, the consumption of phytoestrogen rich foods has been shown to reduce the development of hormone dependent breast cancer. Phytoestrogens improved the efficacy of ongoing chemotherapy. Recent research indicates that polyphenols found in the tamarind seed coat have anti-inflammatory, hepatoprotective, antibacterial and antioxidant activities. The current study was undertaken to evaluate the modulatory potential of methanolic extract of seed coat of T. indica on oestrogen and progesterone production in MCF-7 cancer cell line. T. indica seeds were procured locally, shade dried and the seed coat was removed and extracted with methanol, followed by concentration of the extract using a rotary vacuum evaporator. The qualitative phytochemical analysis of seed coat extract was performed. The cytotoxicity of T. indica seed coat was assessed in MCF-7 cells using MTT assay and the IC50 was determined. The presence of alkaloids, flavonoids, steroids, phenolic compounds, diterpenes, saponins, glycosides and tannins were discovered by qualitative phytochemical analysis. T. indica seed coat decreased cell viability in a dose dependent manner, with an IC50 value of 16 µg/mL. There was dose dependent decrease in oestrogen concentration, whereas the progesterone concentration was found to be increased after 96hrs of treatment with the extract. From the study it could be concluded that methanolic extract of T. indica showed cytotoxicity in vitro against MCF-7 cell lines and it positively modulated progesterone secretion and negatively modulated oestrogen concentration in a time dependent fashion in MCF-7 cell lines. Present in vitro study shows that methanolic extract seed coat of T. indica may have promising role in breast cancer prevention, hence it can be used to develop novel compounds against hormone dependent breast cancer.

In Vitro and In Silico Determination of Some N-ferrocenylmethylaniline Derivatives as Anti-Proliferative Agents Against MCF-7 Human Breast Cancer Cell Lines.

Since the binding of estradiol to its receptor promotes breast cancer cell proliferation (in the ER+ tumours), many molecules targeting this protein have been synthesized to counteract the estradiol action. Ferrocene derivatives have proved their efficiency against hormone-dependent breast cancer cells (MCF-7). In this study, we aimed to find new ferrocene derivatives having pharmacochemistry properties as potential drugs against human breast cancer cells. A series of 29 N-ferrocenylmethylaniline derivatives A0-A28 were synthesised, and their anti-proliferative activity against both hormone-dependent (MCF-7) and independent (MDA-MB 231) human breast cancer cell lines were performed using the MTT test. Molecular docking and drug-likeness prediction were also performed for the five most active derivatives towards MCF-7. A QSAR model was also developed for the perdition of the anti-proliferative activity against MCF-7 cell lines using molecular descriptors and MLR analysis. All studied derivatives demonstrated better cytotoxicity against MCF-7 compared to the MDA-MB-231 cell lines, and compounds A2, A9, A14, A17 and A27 were the most potent ones but still less active than the standard anticancer drug, crizotinib. The QSAR study revealed good predictive ability, as shown by R2 cv = 0.848. In vitro and in silico results indicated that derivatives A2, A9, A14, A17, and A27 possess the highest anti-proliferative activity; these results can be used to design more potent N-ferrocenylmethylaniline derivatives as anti-proliferative agents.

Synthesis, Radiolabeling, and Preclinical Evaluation of 68Ga/177Lu-Labeled Leuprolide Peptide Analog for the Detection of Breast Cancer.

Objectives: The expansion of novel and potent tumor receptor binding peptides is a promising approach for the precise targeting of various cancer. Leuprolide is a 9-residue peptide analog of gonadotropin-releasing hormone and is extensively used in the treatment of sex hormone-dependent tumors, including prostate, breast, and ovarian cancer. This preclinical study was undertaken to prepare a new radiolabeled leuprolide peptide for the detection of breast carcinoma. Methods: A 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-coupled 9-amino acid leuprolide peptide was synthesized after typical 9-fluorenylmethyl-oxycarbonyl-based solid-phase peptide synthesis and radiolabeled with both 68Ga and 177Lu radionuclides for theranostic use. The systemic pharmacokinetics was done in healthy balb/c mice. The in vitro tumor cell binding affinity was determined on MCF7, T47D, and MDA-MB-231 breast cancer cell lines. In vivo tumor targeting and micro positron-emission tomography imaging was performed on nude mice with MCF7 breast tumor xenografts. Results: The leuprolide peptide was conveniently synthesized by solid-phase synthesis strategy and its identity and purity were validated by mass spectrometry and high-performance liquid chromatography. The peptide radiolabeled efficiently (˃94%) with both diagnostic (68Ga) and therapeutic (177Lu) radionuclides and displayed nanomolar binding potency to all three tested MCF7, T47D, and MDA-MB-231 cell lines. Fast and favorable pharmacokinetics was observed for 68Ga/177Lu-leuprolide in healthy Balb/c mice. In nude mice, 68Ga-leuprolide peptide exhibited rapid clearance from the blood circulation with low to moderate (up to 5% ID/g) uptake/retention by the major body organs. The accumulation in the estrogen receptor-positive MCF7 tumor was 2.24% ± 0.62% ID/g at 45 min p.i, with good tumor to blood and muscle uptake ratios. The radiolabeled peptide was excreted primarily through the renal pathway. Conclusion: The encouraging results of this initial study demonstrate that additional testing of this leuprolide peptide seems to be indicated because of its convincing potential to be a new agent for the management of breast carcinoma.

Chloroxylon swietenia (Roxb.) DC induces cell death and apoptosis by down-regulating the NF-κB pathway in MCF-7 breast cancer cells: In vitro and in vivo investigations.

BackgroundNatural products with targeted bioactivity have gained major attention in the field of cancer research owing to emerging anti‐cancer drug resistance and off target toxicities. Chloroxylon swietenia (Roxb.) DC is recognized as a folklore medicinal plant and has numerous therapeutic benefits in the folklore medicine system, however the anti‐cancer potential of this plant and its mechanism of action is poorly understood.AimsThe aim of the study was to investigate the anti‐breast cancer efficacy of C. swietenia leaves methanol extract (CSLME) against MCF‐7 hormone dependent human breast cancer cell line with possible mechanism of action.Methods and resultsThe anti‐breast cancer activity of CSLME against MCF‐7 cells was assessed by evaluating its efficacy toward cytotoxicity, cell migration, colony formation, DNA fragmentation, apoptosis, cytoskeleton, angiogenesis, cell cycle regulation, and animal toxicity. The preliminary screening of CSLME against MCF‐7 cells revealed the cytotoxicity (IC50 20 μg/ml), inhibited cell migration, colony formation, and angiogenesis. It was observed that CSLME induces apoptosis by nuclear fragmentation and disruption of cytoskeleton by actin derangement. The results of Annexin V‐FITC assay and cell cycle analysis by flow cytometry clearly pointed out the sizable fraction of apoptotic cells, and arrested the cells at G2/M phase of cell cycle. The results of the immunoblotting experiments showed that CSLME activates intrinsic pathway of apoptosis with down regulation of anti‐apoptotic marker like Bcl2, up regulation of pro‐apoptotic markers like Bax & Bad, along with successful cleavage of Caspase‐9 and PARP‐1. Further, western blot analysis revealed the possible down regulation of NF‐κB pathway by CSLME, which may be responsible for anti‐cancer activity in MCF‐7 cells. In vivo animal model studies using NOD‐SCID mice demonstrated impressive anti‐tumor activity with significant reduction in tumor volume of MCF‐7 tumor xenograft. Of note, in‐vivo acute oral toxicity study as per Organization for Economic Cooperation and Development 423 revealed the nontoxic nature of CSLME.ConclusionThe in vitro and in vivo findings clearly outline the potential of CSLME as inhibitor of growth and proliferation of MCF‐7 cells. Mechanistically, CSLME seems to activate intrinsic pathway of apoptosis, arrest cell cycle, target actin cytoskeleton, inhibit growth, colony formation, migration, and angiogenesis, with down regulation of NF‐κB pathway leading to cell death.

52P Novel inhibitors of androgen receptor with antiproliferative potency against hormone-sensitive and hormone-resistant breast cancer cells

The growth of most breast cancers depends on estrogens. The hormone therapy is aimed at reducing the effect of estrogens and their receptors on tumor cells and suppressing the rate of tumor growth. The androgen receptor, another steroid receptor, is a novel potential target in breast cancer cells. The work aims to develop selective androgen receptor inhibitors with proapoptotic activity. A series of 1-substituted isatine-5-sulfonamides was synthesized applying Sandmeyer’s methodology and subsequent alkylation of the heterocycle moiety by various benzyl chlorides. The hormone-dependent MCF7 breast cancer cell line was purchased from ATCC. Antiproliferative activity was assessed by the MTT test. The hormone-resistant MCF7/HT subline was obtained by long-term cultivation of MCF7 cells with the active form of tamoxifen, 4-hydroxytamoxifen (HT). Analysis of the antiproliferative activity of the prepared series of new 1-substituted isatin-5-sulfonamides revealed several compounds with low micromolar IC50 values. Compound LCTA-3344 was more active than the reference drug, well-known apoptosis activator 2 (N-(3,4-dichlorobenzyl)isatine). It was determined that isatin-5-sulfonamide LCTA-3344 at micromolar concentrations induces BCL2-dependent apoptosis in MCF7 cells. Moreover, compound LCTA-3344 significantly blocked androgen receptor signaling. Isatin-5-sulfonamide LCTA-3344 in concentrations from 1 to 50 μM caused suppression of the growth of hormone-resistant cell subline MCF7/HT, the IC50 value was 1.4 μM. Interestingly, the hormone-resistant cells did not lose their sensitivity to LCTA-3344 and were more susceptible compared to the parent MCF7 line, for which the IC50 value was 2.6 μM. Thus, a series 1-substituted isatin-5-sulfonamides with high activity against hormone-sensitive breast cancer cells was obtained. The compound LCTA-3344 exhibiting antiproliferative effects against both hormone-sensitive and hormone-resistant breast cancer cells induced apoptosis and significantly inhibited the activity of the androgen receptor. Further in-depth investigation of the anticancer properties of LCTA-3344 is in development.

Impact of Irradiation on the Pharmacokinetics and Biotransformation of Tamoxifen.

BackgroundThe optimal procedure for combining radiotherapy (RT) with tamoxifen treatment is controversial as RT may alter the pharmacokinetics and biotransformation of tamoxifen. The present study investigated this potential interaction by assessing the pharmacokinetics of tamoxifen during concurrent and sequential RT.MethodPlasma tamoxifen concentration was measured in rats with or without RT 2.0 Gy (RT2.0Gy) or 0.5 Gy (RT0.5Gy) with ultra-high-performance liquid chromatography-tandem mass spectrometry after tamoxifen administration (10 mg/kg, p.o., n = 6). Tamoxifen was either administered 1 h after RT (concurrent condition) or 24 h after RT (sequential condition).ResultsPharmacokinetic data analysis demonstrated that the area under the curve (AUC) and half-life of tamoxifen were 2,004 ± 241 h ng/ml and 6.23 ± 1.21 h, respectively, after tamoxifen administration (10 mg/kg, p.o.). The respective conversion rate of 4-hydroxytamoxifen, N-desmethytamoxifen, and endoxifen for tamoxifen metabolism was 20%, 16%, and 5%. The AUC value of tamoxifen in the RT0.5Gy group was 1.5- to 1.7-fold higher than in the sham and RT2.0Gy groups. The relative bioavailability of tamoxifen at concurrent RT0.5Gy and RT2.0Gy groups ranged from 127% to 202% and from 71% to 152%, respectively. The magnitude of endoxifen, which converted from 4-hydroxytamoxifen and N-desmethyltamoxifen, increased 3- to 5-fold in the concurrent RT groups. By contrast, the AUC of tamoxifen decreased by roughly 24% in the sequential RT2.0Gy group. The conversion ratio of endoxifen was four times higher than that in the sequential RT2.0Gy group compared with rats not exposed to RT.ConclusionThe current study provides advanced pharmacokinetic data to confirm the interaction between RT and hormone therapy. Our findings indicate that RT facilitates the metabolism of tamoxifen to active metabolites and thus imply that combination RT-tamoxifen has potential benefits for the treatment of hormone-dependent breast cancer.

Abstract PD1-04: Estrogen-mediated mechanisms in estrogen receptor-positive breast cancer at the single cell level

Abstract Background: Estrogen typically promotes the progression of hormone-dependent breast cancer through activation of estrogen receptor (ER)-α encoded by ESR1. While estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed as an unexpected outcome of aromatase inhibitor (AI)-resistance, the molecular mechanisms have not yet been fully defined. Methods: Characterization of estrogen regulation in the two ER+ breast cancer patient-derived xenograft (PDX) models with opposite responses to estrogen offered us an unprecedented opportunity to assess how 17β-estradiol (E2) modulates ER+ cancer. We succeeded in establishing estrogen-stimulating (SC31) and estrogen-suppressing (GS3) PDX models. In vivo tumor promotion or suppression by estrogen was confirmed through experiments by implanting E2 pellets in mice carrying SC31/GS3, and then single cell analysis was performed using SC31/GS3 tumors. To investigate whether GS3 would change its response to E2, we performed intermittent E2 treatment every 28 days (E2 pellet on/off every 28 days) and whole-genome RNA sequencing using GS3 tumors. Results: SC31 and GS3 behaved oppositely regarding estrogen-mediated tumor growth. Immunohistochemistry indicated that the number of ERα+ cells and Ki-67+ cells were increased in SC31 and decreased in GS3 after E2 treatment, but progesterone receptor+ cells appeared in both SC31 and GS3 after E2 treatment at the protein level. The E2-induced suppression of GS3 involves ERα, not ERβ, which was wild-type and not amplified. Single cell RNA sequencing analysis of these PDXs had revealed that E2 upregulated the expression of estrogen-regulated genes (e.g., PGR and AREG) in both SC31 and GS3. However, E2 treatment induced cell cycle promotion in SC31, while E2 induced cell cycle arrest in GS3. These gene-expression changes occurred in both ESR1+ cells and ESR1- cells, demonstrating for the first time the influence of estrogen on ESR1- cells in ER+breast tumors. This result suggests that 100% ER positivity is not essential for endocrine response. E2 also upregulated a tumor suppressor gene, IL24, only in GS3. More IL24+ cells were ESR1+ and in G1 phase than IL24- cells. Hallmark apoptosis gene sets were upregulated and the hallmark G2M checkpoint gene set was downregulated in IL24+ cells after E2 treatment. After three rounds of intermittent E2 treatment on GS3, an E2 independent growth developed. Approximately 60% of genes in the intermittent E2-treated sample had the same trend as E2-treated samples (mainly ER target genes), in which 40% of the genes behaved similarly to the placebo-treated sample (mainly cell cycle progression genes). Furthermore, lower levels of IL24 were linked to estrogen independence. Conclusions: Estrogen/ERα signaling increases the expression of estrogen-regulated genes, but it can modulate HR+ tumor growth in different manners. While E2 should activate ERα and regulate ESR1+ cells, our findings regarding ESR1- cells were new and suggested crosstalk between ESR1+ cells and ESR1- cells in both estrogen-stimulating and -suppressing ER+ tumors. Furthermore, our studies demonstrate the potential roles of tumor suppressor gene, IL24 in HR+ cancer. Our findings point to the need to identify biomarkers for patients with estrogen-suppressing tumors who can benefit from E2 treatment after AI resistance; measurements of ER and PR expression are insufficient. Expression of IL24 in AI-resistant tumors may be one such indicator for favorable response to E2 therapy. Citation Format: Hitomi Mori, Kohei Saeki, Gregory Chang, Jinhui Wang, Xiwei Wu, Pei-Yin Hsu, Noriko Kanaya, George Somlo, Masafumi Nakamura, Andrea Bild, Shiuan Chen. Estrogen-mediated mechanisms in estrogen receptor-positive breast cancer at the single cell level [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 PD1-04.

The estrogen receptor beta agonist liquiritigenin enhances the inhibitory effects of the cholesterol biosynthesis inhibitor RO 48-8071 on hormone-dependent breast-cancer growth.

Most hormone-dependent human breast cancers develop resistance to anti-hormone therapy over time. Our goal was to identify novel treatment strategies to avoid this drug resistance and thereby control hormone-dependent breast cancer. Sulforhodamine B assays were used to measure viability of cultured human breast-cancer cells. BT-474 cell tumor xenografts in nude mice were used to evaluate tumor growth. Immunohistochemistry was used to assess estrogen-receptor and angiogenesis-marker expression, as well as apoptosis, in tumor-xenograft tissues. MCF-7 and BT-474 breast-cancer cells treated with either RO 48-8071 <[4'-[6-(Allylmethylamino)hexyloxy]-4-bromo-2'-fluorobenzophenone fumarate] [RO]; a small-molecule inhibitor of oxidosqualene cyclase, a key enzyme in cholesterol biosynthesis> or liquiritigenin [LQ; an estrogen receptor (ER) β agonist] exhibited significantly reduced viability in vitro. RO + LQ treatment further significantly reduced cell viability. Administration of RO, LQ, or RO + LQ significantly inhibited growth of BT-474 tumor xenografts in vivo. RO, LQ, or RO + LQ reduced ERα but induced ER β expression in tumor xenografts. Both compounds significantly reduced angiogenesis-marker expression and increased apoptosis in tumor xenografts; use of RO + LQ significantly enhanced the effects observed with a single agent. The ERβ ligand LQ significantly enhanced the inhibition of breast-cancer cell viability and tumor-xenograft growth by RO. The anti-tumor properties of RO may in part be due to an off-target effect that reduces ERα and increases ERβ, the latter of which can then interact with LQ to promote anti-proliferative effects. The RO + LQ combination may have value when considering novel treatment strategies for hormone-dependent breast cancer.

Dose-dependent effects of Hedyotis diffusa extract on the pharmacokinetics of tamoxifen, 4-hydroxytamoxifen, and N-desmethyltamoxifen.

Tamoxifen, a widely prescribed medication in premenopausal women diagnosed with hormone-dependent breast cancer, is potentially co-prescribed with Hedyotis diffusa (H. diffusa), particularly in Taiwan. However, no related report has investigated the drug-herb interaction of H. diffusa on the pharmacokinetics of tamoxifen and its metabolites. In the present study, male Sprague-Dawley rats were administered different doses of H. diffusa extract for 5 consecutive days prior to the administration of tamoxifen (10mg/kg). A validated ultra-liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system was developed to monitor tamoxifen, 4-hydroxytamoxifen, N-desmethyltamoxifen, and endoxifen in rat plasma. Pharmacokinetic results demonstrated that the area under curves (AUCs) of tamoxifen and the relative bioavailability (%) of tamoxifen were dose-dependently decreased (31-68%) by pre-treatment with H. diffusa extract (3g/kg and 6g/kg). In addition, the conversion ratio of 4-hydroxytamoxifen was downregulated (0.5-fold change) and the N-desmethyltamoxifen conversion ratio was upregulated (2-fold change) by high-dose H. diffusa extract. As a result, the relative bioavailability and biotransformation changes affect the clinical efficacy of tamoxifen treatment. These preclinical findings reveal a hitherto unreported interaction between tamoxifen and H. diffusa extract that has implications for their therapeutic efficacy in treating breast cancer.

Influence of Estrogen Treatment on ESR1+ and ESR1- Cells in ER+ Breast Cancer: Insights from Single-Cell Analysis of Patient-Derived Xenograft Models.

Simple SummaryThe benefit of endocrine therapy is normally observed for cancers with 10% or more of cells positive for ER expression. We compared the gene expression profiles in both ESR1+ and ESR1– cells in ER+ tumors following estrogen treatment. Our single-cell RNA sequencing analysis of estrogen-stimulated (SC31) and estrogen-suppressed (GS3) patient-derived xenograft models offered an unprecedented opportunity to address the molecular and functional differences between ESR1+ and ESR1– cells. While estrogen should activate ERα and stimulate ESR1+ cells, our findings regarding ESR1– cells were important, indicating that the proliferation of ESR1– cells in ER+ cancer is also influenced by estrogen. Another valuable finding from our studies was that estrogen also upregulated a tumor-suppressor gene, IL-24, only in GS3. Estrogen increased the percentage of cells expressing IL-24, associated with the estrogen-dependent inhibition of GS3 tumor growth.A 100% ER positivity is not required for an endocrine therapy response. Furthermore, while estrogen typically promotes the progression of hormone-dependent breast cancer via the activation of estrogen receptor (ER)-α, estrogen-induced tumor suppression in ER+ breast cancer has been clinically observed. With the success in establishing estrogen-stimulated (SC31) and estrogen-suppressed (GS3) patient-derived xenograft (PDX) models, single-cell RNA sequencing analysis was performed to determine the impact of estrogen on ESR1+ and ESR1– tumor cells. We found that 17β-estradiol (E2)-induced suppression of GS3 transpired through wild-type and unamplified ERα. E2 upregulated the expression of estrogen-dependent genes in both SC31 and GS3; however, E2 induced cell cycle advance in SC31, while it resulted in cell cycle arrest in GS3. Importantly, these gene expression changes occurred in both ESR1+ and ESR1– cells within the same breast tumors, demonstrating for the first time a differential effect of estrogen on ESR1– cells. E2 also upregulated a tumor-suppressor gene, IL-24, in GS3. The apoptosis gene set was upregulated and the G2M checkpoint gene set was downregulated in most IL-24+ cells after E2 treatment. In summary, estrogen affected pathologically defined ER+ tumors differently, influencing both ESR1+ and ESR1– cells. Our results also suggest IL-24 to be a potential marker of estrogen-suppressed tumors.

Abstract P074: STAT3 pathway in palbociclib resistance in breast cancer cell lines and the role of inhibitors such as tocilizumab and silymarin

Abstract Introduction Resistance to cyclin inhibitors such as palbociclib is a challenge in the treatment of hormone-dependent metastatic breast cancer. Likewise, triple negative tumors are resistant to these inhibitors. The role of the present research is to identify the role that the transcription factor STAT3 (antiapoptotic transcription factor) and the inhibitors of the STAT3 pathway, such as tocilizumab (interleukin 6 receptor inhibitor) and silymarin (extract of milk thistle containing silibinin, a STAT3 inhibitor), could have in this situation. Methods Breast cancer cell lines MCF-7 (positive for estrogen receptors) and MDA-MB-231 (triple negative) were cultured in DMEM + 10% FBS for 72 hours with different concentrations of palbociclib, tocilizumab and silymarin in order to obtain cell growth inhibition values ​​(IC50) in each compound and combinations of palbociclib with tocilizumab or silymarin. In these cases, the combination indexes (CI &amp;lt; 1 (synergy) and CI &amp;gt; 1 (antagonism)) were calculated using CompuSyn software. Likewise, an RNA extraction was carried out from the untreated cell lines to determine the basal expression levels of STAT3 by RT-qPCR. Results The IC50 of palbociclib in MCF-7 was 3.14 µM and in MDA-MB-231, 29.69 µM. The results of RT-qPCR for the basal expression of STAT3 revealed its absence of expression in MCF-7 and the presence of expression in MDA-MB-231, which could be related to the greater resistance to palbociclib of this last cell line. Tocilizumab did not present cytotoxicity per se and silymarin did not reach IC50 value in the cell lines studied (both compounds in doses up to 100 µM). Regarding the combinations of silymarin (50 µM and 75 µM) and palbociclib (0.1 µM and 1 µM), a synergistic effect (CI &amp;lt; 1) was detected in all the combinations studied in MCF-7 and in most of the same in MDA-MB-231 (except 75 µM silymarin and 0.1 µM palbociclib). However, in the case of the combination of tocilizumab (10 µM and 50 µM) and palbociclib (0.1 µM, 1 µM and 5 µM in MCF-7, and 5 µM, 10 µM and 25 µM in MDA-MB-231) synergy was only obtained in half of the combinations studied in both cell lines. Conclusion STAT3 factor could be related to resistance to cyclins in hormone-dependent breast cancer and to the lower sensitivity to these drugs observed in triple-negative breast cancer. The synergistic combination of palbociclib with silymarin, a STAT3 inhibitor, in both the hormone-sensitive and triple negative cell lines indicates that silymarin could help to enhance the effect of palbociclib in breast cancer and, even, help to reverse the resistance to this drug both in the hormone dependent and triple negative, although further studies would be needed to confirm this fact. Citation Format: Kevin Doello, Cristina Mesas, Víctor Amecua, Marco Fuel, Laura Cabeza, Raúl Ortiz. STAT3 pathway in palbociclib resistance in breast cancer cell lines and the role of inhibitors such as tocilizumab and silymarin [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P074.