Breast Cancer Xenograft Model Research Articles

Dynamics-Based Discovery of Novel, Potent Benzoic Acid Derivatives as Orally Bioavailable Selective Estrogen Receptor Degraders for ERα+ Breast Cancer.

The estrogen receptor α (ERα) is identified as an effective target for the treatment of ERα+ breast cancer; thus, discovery of novel selective estrogen receptor degraders (SERDs) are developed as an effective method to overcome the resistance of breast cancer. Herein, the hot-spot residues for protein-ligand interaction between SERDs and ERα are analyzed by molecular dynamic simulation technology, focusing on the hot-spot residues for four series of designed and synthesized SERDs. SAR studies revealed that while the acrylic acid moiety of AZD9496 is scaffold hopping into benzoic acid, compound D24 exhibits potent binding affinity with ERα, good degradation efficacy of ERα, and inhibitory effect against the MCF-7 breast cancer cell line. Besides, D24 also displays good antitumor efficacy in the MCF-7 human breast cancer xenograft model in vivo, favorable pharmacokinetic properties, excellent druggability, and good safety property, making D24 as a promising drug candidate of SERD for further evaluation.

Neratinib plus trastuzumab is superior to pertuzumab plus trastuzumab in HER2-positive breast cancer xenograft models

Lapatinib (L) plus trastuzumab (T), with endocrine therapy for estrogen receptor (ER)+ tumors, but without chemotherapy, yielded meaningful response in HER2+ breast cancer (BC) neoadjuvant trials. The irreversible/pan-HER inhibitor neratinib (N) has proven more potent than L. However, the efficacy of N+T in comparison to pertuzumab (P) + T or L + T (without chemotherapy) remains less studied. To address this, mice bearing HER2+ BT474-AZ (ER+) cell and BCM-3963 patient-derived BC xenografts were randomized to vehicle, N, T, P, N+T, or P+T, with simultaneous estrogen deprivation for BT474-AZ. Time to tumor regression/progression and incidence/time to complete response (CR) were determined. Changes in key HER pathway and proliferative markers were assessed by immunohistochemistry and western blot of short-term-treated tumors. In the BT474-AZ model, while all N, P, T, N + T, and P + T treated tumors regressed, N + T-treated tumors regressed faster than P, T, and P + T. Further, N + T was superior to N and T alone in accelerating CR. In the BCM-3963 model, which was refractory to T, P, and P + T, while N and N + T yielded 100% CR, N + T accelerated the CR compared to N. Ki67, phosphorylated (p) AKT, pS6, and pERK levels were largely inhibited by N and N + T, but not by T, P, or P + T. Phosphorylated HER receptor levels were also markedly inhibited by N and N + T, but not by P + T or L + T. Our findings establish the efficacy of combining N with T and support clinical testing to investigate the efficacy of N + T with or without chemotherapy in the neoadjuvant setting for HER2+ BC.

Therapeutic Uses of Bacterial Subunit Toxins.

The B subunit pentamer verotoxin (VT aka Shiga toxin-Stx) binding to its cellular glycosphingolipid (GSL) receptor, globotriaosyl ceramide (Gb3) mediates internalization and the subsequent receptor mediated retrograde intracellular traffic of the AB5 subunit holotoxin to the endoplasmic reticulum. Subunit separation and cytosolic A subunit transit via the ER retrotranslocon as a misfolded protein mimic, then inhibits protein synthesis to kill cells, which can cause hemolytic uremic syndrome clinically. This represents one of the most studied systems of prokaryotic hijacking of eukaryotic biology. Similarly, the interaction of cholera AB5 toxin with its GSL receptor, GM1 ganglioside, is the key component of the gastrointestinal pathogenesis of cholera and follows the same retrograde transport pathway for A subunit cytosol access. Although both VT and CT are the cause of major pathology worldwide, the toxin–receptor interaction is itself being manipulated to generate new approaches to control, rather than cause, disease. This arena comprises two areas: anti neoplasia, and protein misfolding diseases. CT/CTB subunit immunomodulatory function and anti-cancer toxin immunoconjugates will not be considered here. In the verotoxin case, it is clear that Gb3 (and VT targeting) is upregulated in many human cancers and that there is a relationship between GSL expression and cancer drug resistance. While both verotoxin and cholera toxin similarly hijack the intracellular ERAD quality control system of nascent protein folding, the more widespread cell expression of GM1 makes cholera the toxin of choice as the means to more widely utilise ERAD targeting to ameliorate genetic diseases of protein misfolding. Gb3 is primarily expressed in human renal tissue. Glomerular endothelial cells are the primary VT target but Gb3 is expressed in other endothelial beds, notably brain endothelial cells which can mediate the encephalopathy primarily associated with VT2-producing E. coli infection. The Gb3 levels can be regulated by cytokines released during EHEC infection, which complicate pathogenesis. Significantly Gb3 is upregulated in the neovasculature of many tumours, irrespective of tumour Gb3 status. Gb3 is markedly increased in pancreatic, ovarian, breast, testicular, renal, astrocytic, gastric, colorectal, cervical, sarcoma and meningeal cancer relative to the normal tissue. VT has been shown to be effective in mouse xenograft models of renal, astrocytoma, ovarian, colorectal, meningioma, and breast cancer. These studies are herein reviewed. Both CT and VT (and several other bacterial toxins) access the cell cytosol via cell surface ->ER transport. Once in the ER they interface with the protein folding homeostatic quality control pathway of the cell -ERAD, (ER associated degradation), which ensures that only correctly folded nascent proteins are allowed to progress to their cellular destinations. Misfolded proteins are translocated through the ER membrane and degraded by cytosolic proteosome. VT and CT A subunits have a C terminal misfolded protein mimic sequence to hijack this transporter to enter the cytosol. This interface between exogenous toxin and genetically encoded endogenous mutant misfolded proteins, provides a new therapeutic basis for the treatment of such genetic diseases, e.g., Cystic fibrosis, Gaucher disease, Krabbe disease, Fabry disease, Tay-Sachs disease and many more. Studies showing the efficacy of this approach in animal models of such diseases are presented.

A small molecule LN435a targeting TGFβR1 exerts promising antitumor effects on breast cancer.

e15069 Background: Breast cancer has overtaken lung cancer as the most diagnosed cancer. Despite conventional treatment, metastases occur in 20-30% of patients, resulting in death. This study aims to screen of effective drugs by metastatic patient-derived organoid and the potential molecular mechanism. Methods: Breast Cancer patient-derived organoid (PDO) model was established from the patient who have multiple drug resistance, multiple visceral and contralateral breast metastases. The organoid morphologies was tested by hematoxylin-eosin (HE) staining and immunohistochemistry (IHC). Then, pharmacological activity assay of 2370 natural product monomer (from Selleck) was performed with organoids. we modified the structure of harmine(HM) and screened the best active drugs. Cell proliferation assay and wound healing assay were used to detect LN435a anticancer activity in vitro. Orthotopic, Metastatic Xenograft and Patient-Derived tumor Xenograft(PDX) model of Breast Cancer were used to detect LN435a anticancer activity in vivo. In order to explore the anti-cancer target of LN435a, we used RNA transcriptome and proteomics sequencing. To further validate anti-cancer targets,TGFβ receptor 1 (TGFβR1), we used real-time quantitative qPCR, western blot, lentiviral packing and biolayer interferometry assay. To investigate whether LN435a inhabition of EMT and stem cell markers, we performed flow cytometry, immunohistochemistry and fluorescence. Results: We observe that organoid morphologies typically matched the histopathology, hormone receptor status, and HER2 status of the original tumor. In the first anti-cancer drug screening, HM showes the best effect on PDO. Because HM contains β-carbine alkaloids as the structural units, we designe a series of active drugs based on this and did anticancer screening. We find LN435a as one of the lead compounds exerting anti-metastatic activity in the nanomolar range in PDO and breast cancer cells. Proteomic and biochemical studies identify TGFβR1 as the direct target of LN435a. And then it inhibits EMT and stem cell markers. In parallel, loss of TGFβR1 or pharmacological inhibition of TGFβR1 by LN435a reduces breast cancer extravasation into the lung in an experimental metastasis mouse model, which reveals an essential role of TGFβR1 in breast cancer progression. Conclusions: Altogether, LN435a is a novel inhibitor of promising anti-tumor effects on breast cancer that works by blocking TGFβ signaling pathways.

Bispecific Estrogen Receptor α Degraders Incorporating Novel Binders Identified Using DNA-Encoded Chemical Library Screening

Bispecific degraders (PROTACs) of ERα are expected to be advantageous over current inhibitors of ERα signaling (aromatase inhibitors/SERMs/SERDs) used to treat ER+ breast cancer. Information from DNA-encoded chemical library (DECL) screening provides a method to identify novel PROTAC binding features as the linker positioning, and binding elements are determined directly from the screen. After screening ∼120 billion DNA-encoded molecules with ERα WT and 3 gain-of-function (GOF) mutants, with and without estradiol to identify features that enrich ERα competitively, the off-DNA synthesized small molecule exemplar 7 exhibited nanomolar ERα binding, antagonism, and degradation. Click chemistry synthesis on an alkyne E3 ligase engagers panel and an azide variant of 7 rapidly generated bispecific nanomolar degraders of ERα, with PROTACs 18 and 21 inhibiting ER+ MCF7 tumor growth in a mouse xenograft model of breast cancer. This study validates this approach toward identifying novel bispecific degrader leads from DECL screening with minimal optimization.

Preclinical meritorious anticancer effects of Metformin against breast cancer: An In vivo trial

ObjectiveThis research aims to evaluate the preclinical meritorious and anticancer effects of Metformin in a Xenograft model of breast cancer. MethodsThis interventional trial was conducted during a defined period of 5 months (August 2016 January 2017). We used a Xenograft model of nude BALB/c mice. A sample size of 50 mice, allocated into two groups and designated as Group A and Group B for Metformin and negative control groups, respectively. The anticancer activity of Metformin has been evaluated by comparing the tumour volume, tumour weight, tumour regression ratio, percentage regression, and survival rate. ResultsCompared with the control group, Metformin can significantly reduce the progression of tumour in the Xenograft model of breast cancer induced by MCF-7. This is reflected by significant differences in tumour volume at the final follow-up (p = <0.001). Our findings are further supported by a significant reduction of the tumour growth rate (p = <0.001) and tumour weight (p = <0.001) in the Metformin group than in the control group. Similarly, the total survival rate and tumour regression are more significantly correlated in the Metformin group. ConclusionThis study demonstrates that Metformin can significantly reduce the tumour growth and can increase the survival rate in a Xenograft model of breast cancer.

Tumor co-expression of progranulin and sortilin as a prognostic biomarker in breast cancer

BackgroundThe growth factor progranulin has been implicated in numerous biological processes such as wound healing, inflammation and progressive tumorigenesis. Both progranulin and its receptor sortilin are known to be highly expressed in subgroups of breast cancer and have been associated with various clinical properties including tamoxifen resistance. Recent data further suggest that progranulin, via its receptor sortilin, drives breast cancer stem cell propagation in vitro and increases metastasis formation in an in vivo breast cancer xenograft model. In this retrospective biomarker analysis, we aimed to determine whether tumor co-expression of progranulin and sortilin has prognostic and treatment predictive values for breast cancer patients.MethodsWe explored how co-expression of progranulin and sortilin was associated with established clinical markers by analyzing a tissue microarray including 560 randomized premenopausal breast cancer patients receiving either 2 years of tamoxifen treatment or no adjuvant treatment, with a median follow-up time of 28 years. Breast cancer-specific survival was analyzed using Kaplan-Meier and Cox Proportional Hazards regression models to assess the prognostic and predictive value of progranulin and sortilin in relation to known clinical markers.ResultsCo-expression of progranulin and sortilin was observed in 20% of the breast cancer samples. In untreated patients, prognostic considerations could be detailed separately from treatment prediction and the high progranulin and sortilin expressing subgroup was significantly associated with breast cancer-specific death in multivariable analyses (HR=2.188, CI: 1.317–3.637, p=0.003) along with tumor size, high tumor grade and lymph node positivity. When comparing the untreated patients with tamoxifen treated patients in the ERα positive subgroup, co-expression of progranulin and sortilin was not linked to tamoxifen resistance.ConclusionData suggest that co-expression of progranulin and its receptor sortilin is a novel prognostic biomarker combination identifying a highly malignant subgroup of breast cancer. Importantly, this subpopulation could potentially be targeted with anti-sortilin based therapies.

Abstract PS18-26: Men1611 promotes immune activating myeloid cell polarization

Abstract MEN1611 is a PI3K inhibitor currently in clinical development targeting the p110 α, β and γ isoforms, while sparing the δ subunit. In preclinical models, MEN1611 has demonstrated a long lasting antitumor activity when combined with trastuzumab in HER2+/PiK3CA mutated breast cancer. Aim of this work is to characterize the effects of MEN1611 on the PI3Kγ isoform, highly expressed in tumor-associated macrophages (TAMs), in order to understand whether the anti-tumor activity of MEN1611 might be also mediated by the inflammatory microenvironment. Previous evidences have shown that selective targeting of PI3Kγ by IPI-549 can reshape the inflammatory cells infiltrating tumors towards a less immunosuppressive phenotype and promote cytotoxic T cell-mediated tumor regression. In order to evaluate the effect of MEN1611 on TAMs, we established an in vitro model by differentiating murine and human macrophages from bone marrow-derived monocytes or buffy coats-isolated monocytes respectively. Pro-inflammatory M1 macrophages have been differentiated by treatment with Lipopolysaccharide (LPS) and interferon γ (IFNγ), while pro-tumoral M2 macrophages by interleukin 4 (IL-4) stimulation. The cells, thus obtained, have been incubated with MEN1611 or IPI-549 (positive control) and the effects on their phenotype, gene and protein expression, evaluated through confocal microscopy, RNASeq and flow cytometry respectively. A syngeneic xenograft model of breast cancer based on 4T1 cells has been also established in order to investigate in vivo the activity of MEN1611 on the inflammatory environment (TAMs and T cells). In vitro data showed MEN1611 and IPI-549 ability to repolarize murine and human macrophages towards a pro-inflammatory phenotype. Confocal analysis revealed a shape remodeling from an elongated M2-like towards a round M1-like; gene and protein expression analysis revealed a significant increase of immuno-stimulating factors mRNA and M1 chemokines and cytokines secretion, respectively. A modulation of the inflammatory infiltrate was also observed in vivo where RNASeq and flow cytometry analysis on dissociated treated tumors highlighted an increase of pro-inflammatory markers. The in-silico ingenuity pathway analysis (IPA) performed on genes modulated by MEN1611 revealed the enhancement of some processes related to an immune activating switch, such as the recruitment of myeloid cells or the cytotoxicity of lymphocytes and natural killer cells. In conclusion, we demonstrated in a cellular context that MEN1611 activity on PI3Kγ isoform is responsible for macrophages reprogramming from an immune-suppressive to an immune-activating phenotype. Moreover, we observed that in 4T1 murine breast cancer model, tumor regression induced by MEN1611 was also sustained by a modulation of the inflammatory microenvironment, characterized by an increased recruitment and cytotoxicity of T cells. Citation Format: Stefania Capano, Alessandro Paoli, Alessio Fiascarelli, Andrea Massimiliano Tomirotti, Mario Bigioni, Alessandro Bressan, Daniela Bellarosa, Massimiliano Salerno, Monica Binaschi. Men1611 promotes immune activating myeloid cell polarization [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-26.

Abstract OT-09-02: A randomized, open-label, parallel-group, multicenter phase 2 study comparing the efficacy and safety of oral AZD9833 versus fulvestrant in women with advanced ER-positive HER2-negative breast cancer (SERENA-2)

Abstract Background AZD9833 is a next-generation oral selective estrogen receptor (ER) antagonist and degrader (SERD) that has shown anti-tumor efficacy in a range of pre-clinical xenograft models of breast cancer. The first-in-human study, assessing AZD9833 as a monotherapy and in combination with palbociclib (SERENA-1; NCT03616587), established a dose-dependent safety profile with clinical benefit and target engagement in pre- and post-menopausal women at all dose levels. Here, we describe the design of SERENA-2, a Phase 2 randomized, open-label trial of three different doses of AZD9833 versus fulvestrant. Methods SERENA-2 is a global comparative study of three different doses of AZD9833 versus fulvestrant in post-menopausal women with advanced ER+, HER2− breast cancer with disease recurrence or progression after ≥1 endocrine therapy. The study will evaluate the efficacy and safety of AZD9833 monotherapy once daily at three dose levels, versus fulvestrant monotherapy administered according to its label. Eligible patients will have received no prior fulvestrant or other oral SERD, and no more than one endocrine therapy and one chemotherapy in the advanced setting. Prior treatment with CDK4/6 inhibitors is permitted. Patients will be randomized 1:1:1:1 to one of four treatment groups: AZD9833 75 mg, 150 mg, 300 mg, or fulvestrant. The primary objective of the study is to determine the clinical efficacy of AZD9833 as assessed by progression-free survival, compared with fulvestrant. Secondary objectives include objective response rate, duration of response, percentage change in tumor size at 16 weeks, clinical benefit rate at 24 weeks, and overall survival. Pharmacokinetics, pharmacodynamic biomarker changes from baseline, and effects of AZD9833 and fulvestrant on patients’ health-related quality of life will also be assessed. Exploratory endpoints include predictive markers of response and/or acquired resistance to AZD9833 and fulvestrant, including circulating tumor DNA mutation profiling and dynamics, circulating tumor cell enumeration, and analysis of tumor samples. Patient enrollment commenced in Q2 2020, with a target enrollment of 288 patients across approximately 100 sites in up to 17 countries. Efficacy analyses will compare each dose of AZD9833 with fulvestrant. Sample size was calculated to provide 80% power for the primary endpoint. The primary analysis will use a Cox proportional hazards model stratified by prior use of CDK4/6 inhibitors and presence of lung and/or liver metastases to compare progression-free survival in each dose of AZD9833 versus fulvestrant. Another randomized, open-label, parallel-group, pre-surgical study investigating the biological effects of AZD9833 in ER+, HER2− primary breast cancer (SERENA-3) is also ongoing. For more information please contact Dr Mafalda Oliveira at: moliveira@vhio.net. Citation Format: Mafalda Oliveira, Maxine Bennett, Ali Khalil, Richard Mather, Rhiannon Maudsley, Sam McGuinness, Christopher J Morrow, Andy Sykes, Li Zhang, Teresa Klinowska, Justin PO Lindemann, Danielle Carroll. A randomized, open-label, parallel-group, multicenter phase 2 study comparing the efficacy and safety of oral AZD9833 versus fulvestrant in women with advanced ER-positive HER2-negative breast cancer (SERENA-2) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr OT-09-02.

Abstract PS16-22: Targeting resistance to current CDK4/6 therapies by RGT-419B, an inhibitor with optimized kinase activity spectrum

Abstract Cyclin-dependent kinases (CDKs) 4/6 inhibitors are a powerful class of therapeutic drugs for the treatment of advanced metastatic breast cancer. However, the currently approved CDK4/6 inhibitors palbociclib, ribociclib and abemaciclib have dose-limiting toxicities that require treatment holidays or reductions to sub-optimal doses, thus limiting full target inhibition. The residual CDK4/6 activity, together with persistent signaling through CDK2/cyclin E are among key resistant mechanisms that can compromise full clinical benefit. RGT-419B is a 3rd generation CDK inhibitor with an optimized kinase activity spectrum that has been discovered employing a Computer Accelerated Rational Design technology platform. RGT-419B has potent sub-nM CDK4 activity with desired degrees of selectivity against kinases such as CDK6, CDK9 and GSK3β to enable full target engagement with an improved safety profile. Furthermore, single digit nM CDK2 kinase activity has been incorporated into the design of RGT-419B to combat Cyclin E/CDK2-driven resistance. In vitro, RGT-419B showed more robust activity against palbociclib-resistant ER+ breast cancer cells than abemaciclib. In ER+ T47D breast cancer cells with overexpression of Cyclin E1, RGT-419B exhibited better antiproliferation activity than either abemaciclib or palbociclib. RGT-419B also demonstrated more durable in vivo tumor growth inhibition when compared with abemaciclib in an ER+ breast cancer xenograft model. The optimized kinase activity spectrum of RGT-419B provides an opportunity to treat ER+ breast cancer patients refractory to the existing CDK4/6 inhibitors, as either a single agent or in combination with other therapies. Citation Format: Zhi Xie, Jing Han, Zhilong Hu, Hu He, Xianqiang Sun, Fei Zhang, Jing Lin, Lili Yao, Xinjuan Wang, Liufeng Mei, Yangyang Liu, Guoyun Zhu, Xi Chen, Xiaotian Zhu, Wenge Zhong. Targeting resistance to current CDK4/6 therapies by RGT-419B, an inhibitor with optimized kinase activity spectrum [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-22.

Abstract PS11-26: A phase 1 study of D-0502, an orally bioavailable SERD, for advanced or metastatic HR-positive and HER2-negative breast cancer

Abstract Background: Targeting the estrogen receptor (ER) has proven to be one of the most successful strategies in treating HR+ (ER+ and PR+) and HER2- breast cancer. Selective estrogen receptor degraders (SERD) can be used as single agents or in combination with CDK4/6 inhibitors such as palbociclib. Fulvestrant is currently the only approved agent in its class and is limited by poor oral bioavailability necessitating monthly intramuscular injections. D-0502 is an orally bioavailable SERD with potent activity in various HR+ and HER2- breast cancer cell lines and xenograft models. Its combination with palbociclib in both MCF-7 xenograft models and ESR-1 mutated (Y537S) patient derived breast cancer xenograft models resulted in further tumor growth inhibition or regression. Drug metabolism and pharmacokinetic studies both in vitro and in vivo demonstrated that D-0502 exhibits favorable PK profiles suitable for clinical development. Methods: A first-in-human phase 1 study was conducted to evaluate D-0502 in women with advanced or metastatic HR+, HER2- breast cancer (MBC) (NCT03471663). The primary objective is to characterize the safety and tolerability of D-0502 and D-0502 in combination with palbociclib, to identify an MTD and/or RP2D. The secondary objectives are to evaluate the PK properties and the preliminary anti-tumor activities. Patients received D-0502 orally once daily in 28-day cycles. The study has completed dose escalation (phase 1a), and dose expansion and combination studies (phase 1b) are ongoing. In phase Ia, patients were enrolled and evaluated using conventional 3+3 dose-escalation to identify the MTD of D-0502 as a single agent. The phase 1b was divided into 2 stages. In Stage 1, D-0502 was evaluated with palbociclib at a dose below the MTD first before escalating to the MTD. Stage 1 also included patients in China treated at a dose below and at the MTD as single agent as well as in combination with palbociclib. Stage 2 will further evaluate the MTD for both single agent and combination of D-0502 with palbociclib. Results: At the time of this abstract submission, phase 1a dose escalation is completed, R2PD has been identified, and no DLTs were observed. PK analysis of D-0502 indicates a dose proportional increase in exposure as the dose increases, and the exposure has exceeded the potential therapeutic exposure level based on preclinical studies. The Phase Ib Stage 1 portion has also completed enrollment and the study is currently enrolling patients into phase Ib Stage 2. D-0502 as a single agent or in combination has been well tolerated with radiological tumor response and clinical benefit response (CBR) observed. Safety, PK and preliminary efficacy data will be reported at the meeting presentation. Conclusion: A first-in-human phase 1 study of D-0502 has been initiated in women with advanced or metastatic HR-positive and HER2-negative breast cancer. D-0502 has been well tolerated and achieved significant exposure and preliminary clinical activity in patients. Further results will be presented at the meeting. Citation Format: Cynthia Osborne, Donald A Richards, Sharon T Wilks, Sami Diab, Dejan Juric, Kate Lathrop, Andrea Silber, William Edenfield, Amardeep Aulakh, Ben Cho, Binghe Xu, Tao Sun, Quchang Ouyang, Yanxia Shi, Kathryn Stazzone, Zhe Shi, Ling Zhang, Yaolin Wang, Erika P Hamilton. A phase 1 study of D-0502, an orally bioavailable SERD, for advanced or metastatic HR-positive and HER2-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS11-26.

Abstract PS16-07: Blocking H3K27me3 methyltransferase or demethylase activity impacts epithelial-mesenchymal plasticity, suppressing tumor growth and metastasis

Abstract Background: Epithelial-mesenchymal plasticity (EMP) is key feature driving breast cancer metastasis. Induction of epithelial-mesenchymal transition (EMT) increases the migratory and invasive capabilities associated with metastatic competence and endows tumors with stem cell properties necessary for initiating tumor growth in the metastatic site. Mesenchymal-epithelial transition (MET) has been increasingly recognized as an integral part of the latter stages of the metastatic cascade as it is required for re-epithelialization, proliferation, and expansion of micro-metastases into macro-metastases with a histopathology similar to that of the primary tumor. Interconversion between epithelial and mesenchymal states requires remodeling of the epigenome–including addition and erasure of H3K27me3, especially near gene promoter and enhancers. Deposited by EZH1 or EZH2, members of the PRC2 complex, H3K27me3 is associated with gene silencing. However, gene activation occurs upon H3K27me3 demethylation to H3K27me1, mediated by KDM6A or KDM6B, members of the Compass-like complex. Results: Based upon our prior work, which showed EMT-driven re-distribution of H3K27me3 onto and off of genes that are critical regulators of cell fate and differentiation, we asked whether inhibition of H3K27me3-directed methyltransferases and demethylases could impact breast cancer. We found that the EZH2 inhibitor, EPZ011989, reduced EMP by preventing recovery of epithelial trails in cells exposed to TGFβ, but did not, on its own, induce EMT. However, we found that either inhibition of the H3K27me3-directed demethylases, KDM6A and KDM6B, using GSK-J4 or shRNA knockdown, induced EMT and promoted cellular migration. Thus, manipulation of either EZH2 or KDM6A/B affected the capacity of cells to repress or activate expression of genes known to exhibit dynamic gain or loss of H3K27me3 during EMT. Given the effects of inhibitors of H3K27-targeted enzymes on EMP, we sought to ascertain their impact on tumor growth and metastasis. Using a GFP-expressing patient-derived xenograft (PDX) model of ER-positive breast cancer, we observed a dose-dependent suppression of primary tumor growth and fewer disseminated tumor cells (DTCs) in response to small molecules targeting H3K27me3 regulators. Conclusions: These results indicate that addition and removal of H3K27me3 is critical for maintenance of cell fate and plasticity. Furthermore, targeting this pathway suppresses breast cancer growth and metastasis in a mouse model of ER-positive breast cancer. Citation Format: Joseph Taube, Provas Das, Kelsey Johnson, John Landua, Lacey Dobrolecki, Michael Lewis. Blocking H3K27me3 methyltransferase or demethylase activity impacts epithelial-mesenchymal plasticity, suppressing tumor growth and metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-07.

Abstract PS5-40: CDKN2A loss can be a predictive marker of palbociclib in breast and gastric cancer

Abstract Background: Abnormal cell cycle progression is a characteristic of cancer, and targeting the cell cycle is a strategy for cancer treatment. TCGA reported that 7% and 12% of gastric cancers exhibit CCND1 or CCNE1 alterations, respectively. Besides, Cyclin D1 is overexpressed in 25% to 60% of invasive breast carcinomas, and gene amplification is observed in 10% to 30% of breast cancer cases. Furthermore, CDK4/6 and CDNK2A/B aberrations are frequently observed in gastric (39.6%) and breast (7.6%) cancers. The presence of such abnormalities in cell cycle-related molecules suggests that gastric and breast cancers are good candidates for treatment with cell cycle inhibitors. Palbociclib is a specific inhibitor of CDK4/6, a vital regulator of the G1 checkpoint, and has been approved by the FDA because it provided a significant benefit by extending PFS in a phase III trial for hormone-positive advanced breast cancer. However, the predictive marker of palbociclib is not determined. Even though CDKN2A loss has been considered as a sensitive marker of palbociclib, there is no preclinical evidence to support whether CDKN2A deficient cancer shows sensitivity to palbociclib, especially in gastric and breast cancer. Therefore, we investigated the effects of palbociclib on CDKN2A loss gastric and breast cancer cell lines as well as patient derived-xenograft (PDX) models. Methods: The cytotoxic assay, cell cycle analysis, and western blotting were conducted to determine the anti-tumor effect and action mechanisms of palbociclib on gastric and breast cancer cell lines. Moreover, modulation of CDKN2A expression was conducted by siRNA and plasmid overexpression. These in vitro data were validated in vivo model and gastric cancer PDX models which have CDKN2A loss as well. Results: There is a meaningful correlation between CDKN2A loss and palbociclib sensitivity among gastric and breast cancer cell lines. CDKN2A loss cells showed G1 cell cycle arrest by blocking Rb phosphorylation and inhibited proliferative cell signaling. Moreover, palbociclib promoted senescence rather than apoptosis. The depletion of CDKN2A expression using siRNA increased palbociclib sensitivity with G1 cell cycle arrest accompanied by senescence. In contrast, CDKN2A overexpression in sensitive cells showed insensitivity to palbociclib. The anti-tumor effects of palbociclib on CDKN2A loss breast cancer cells were validated in the xenograft model, and the two different gastric cancer PDX models have CDKN2A loss also showed a significant response to palbociclib as well. Conclusions: CDK4/6 inhibitor palbociclib showed an anti-tumor effect in vitro and in vivo xenograft model of CDKN2A loss gastric and breast cancer. Our results suggest that palbociclib has therapeutic potential for the treatment of not only breast cancer but also gastric cancer, not limited to a hormone-positive breast cancer type. Our results provide a rationale for the future clinical trials of palbociclib in the treatment of breast cancers. Citation Format: Ahrum Min, Yu Jin Kim, Miso Lee, Kyung-Hun Lee, Seock-Ah Im. CDKN2A loss can be a predictive marker of palbociclib in breast and gastric cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-40.

Abstract PS8-13: Silicone implant based sustained localized drug delivery of fulvestrant to prevent breast cancer

Abstract Background: Hereditary breast cancer is common. With enhanced awareness and the recent introduction in affordable multi-gene germ line testing, an estimated 0.5-1 million women will learn that they carry a considerable risk to develop breast cancer. Thus, a rapidly increasing number of women, many of them very young, will be in need of effective strategies for breast cancer prevention. Current options to prevent breast cancer in women at high risk include bilateral mastectomies or systemic anti-estrogen therapy. Both options, while effective, may have a detrimental impact on the physical and emotional well-being of the patient. Localized delivery of an established anti-estrogen to breast tissue only may thus offer an attractive alternative for cancer prevention and may replace systemic therapy for ductal carcinoma in situ and early stage breast cancer with minimal risk for metastases. Methods: As such, we have sought to develop a silastic-silicone device, which when placed under breast tissue, will deliver the anti-estrogen fulvestrant directly to the target tissue. Sustained slow release of fulvestrant from a silastic-silicone device directly into the mammary tissue will provide the risk reducing benefits of systemic hormonal therapy while minimizing systemic exposure and the resulting poor compliance due to adverse effects. Results: Using a combination of in vitro and in vivo studies, we show that fulvestrant can be delivered through a silastic-silicone device. Implanted adjacent to mammary tissue, this drug delivery device provides sustained high levels of fulvestrant to inhibit estrogen receptor signaling and breast cancer cell proliferation. In a MCF-7 breast cancer xenograft model we have shown that silastic-silicone delivers fulvestrant selectively to mouse mammary tissue for more than 1 year with anti-tumor effects similar to those achieved with systemic fulvestrant exposure. Using the Sprague-Dawley rat DMBA spontaneous breast cancer induction model, we further demonstrate that fulvestrant delivered by silastic-silicone devices implanted adjacent to mammary tissue significantly delays time to first tumor compared to control animals (n=90, HR = 0.42, 95% CI 0.21-0.83) with minimal systemic exposure (plasma average 1.1 ng/mL, SD ±1.5 ng/mL). Initial large animal safety and toxicity studies in female sheep (ewes, n=2) support the surgical strategy to place the device between breast tissue (i.e. udder) and the chest wall (abdominal wall in ewes). Following 1 month of fulvestrant release, gross and histopathological analysis found no adverse effects or implant related toxicity. Bioanalytical analysis of mammary tissues suggested that drug was found highest near the implant (max 341 ng/g, udder average 58.4 ng/g, SD ±17.1 ng/g), with diminishing levels distal from the implant. Systemic exposure was low, with plasma levels 1.2 ng/mL, SD ±0.22 ng/mL, comparing favorably to clinical plasma levels achieved by intramuscular fulvestrant of 500 mg: 28.0 ng/mL, SD ±27.9 ng/mL (FDA package insert). Subsequent large animal studies will focus on the safety and pharmacokinetics of device delivered fulvestrant in longer studies (e.g. 3 to 6 months). Conclusions: The greater awareness and genetic identification of individuals at risk for breast cancer brings about an increased need for novel approaches to breast cancer prevention. The development of a silastic-silicone based device for sustained and localized drug delivery with an approved and effective anti-estrogen should allow rapid transition into clinical testing. This strategy will provide an alternative option to mastectomies and allow breast conservation for women identified to have a more than 40% lifetime chance of developing breast cancer, as well as provide an alternative to systemic hormonal therapy in women with ductal carcinoma in situ or stage I breast cancer. Citation Format: Pamela N Munster, Pujan K Desai, Nela Pawlowska, Elysia Roche, Romain Pacaud, Maxim A Daud, Emily Hsu, David Gingrich, Amelia Deitchman, Fran Aweeka, Scott Thomas. Silicone implant based sustained localized drug delivery of fulvestrant to prevent breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS8-13.

Glutathionylation-dependent proteasomal degradation of wide-spectrum mutant p53 proteins by engineered zeolitic imidazolate framework-8

Point mutations within the DNA-binding domain of the TP53 gene occur in a significant percentage of human cancer, leading to cellular accumulation of highly stabilized mutant p53 proteins (mutp53) with tumor-promoting properties. Depletion of mutp53, through inducing either autophagic or proteasomal degradation, is an attractive strategy for the therapy of p53-mutated cancer, but the currently-known degradation inducers, almost exclusively small molecules, are inadequate. Here we show that pH-responsive zeolitic imidazolate framework-8 (ZIF-8) offers a novel solution to mutp53 degradation. ZIF-8 facilitated ubiquitination-mediated and glutathionylation-dependent proteasomal degradation of all of the nine mutp53 we tested, including six hot-spot mutp53, but not the wild-type p53 protein. Sustained elevation of intracellular Zn++ level, resulted from decomposition of the internalized ZIF-8 in the acidic endosomes, decreased the intracellular reduced glutathione (GSH): oxidized glutathione (GSSG) ratio and was essential for mutp53 glutathionylation and degradation. ZIF-8 modified with an Z1-RGD peptide, exhibiting enhanced cellular internalization and improved decomposition behavior, preferentially killed mutp53-expressing cancer cells and demonstrated remarkable therapeutic efficacy in a p53 S241F ES-2 ovarian cancer model as well as in a p53 Y220C patient-derived xenograft (PDX) breast cancer model. The ability to induce wide-spectrum mutp53 degradation gives ZIF-8 a clear advantage over other degradation-inducers, and engineered nanomaterials may be promising alternatives to small molecules for the development of mutp53-targeting drugs.

Oral Delivery of Gambogenic Acid by Functional Polydopamine Nanoparticles for Targeted Tumor Therapy

To enhance the water solubility, oral bioavailability, and tumor targeting of gambogenic acid (GNA), polydopamine nanoparticles (PDA NPs) were prepared to encapsulate and stabilize GNA surface modified by folic acid (FA) and then coated with sodium alginate (GNA@PDA-FA SA NPs) to achieve an antitumor effect by oral administration. GNA@PDA-FA SA NPs exhibited in vitro pH-sensitive release behavior. In vitro cell studies manifested that GNA@PDA-FA NPs had higher cytotoxicity to 4T1 cells compared with raw GNA (IC50 = 2.58 μM vs 7.57 μM). After being modified with FA, GNA@PDA-FA NPs were taken up easily by 4T1 cells. In vivo studies demonstrated that the area under the curve (AUC0→∞) of the plasma drug concentration-time of GNA@PDA-FA SA NPs was 2.97-fold higher than that of raw GNA, along with improving drug distribution in the liver, lung, and kidney tissues. In vivo anti-tumor experiments, GNA@PDA-FA SA NPs significantly inhibited the growth of breast tumors in the 4T1 xenograft breast cancer model via oral administration without obvious toxicity on major organs. Our studies indicated that the GNA@PDA-FA SA NPs modified with FA and coated with SA were a promising drug delivery system for targeting tumor therapy via oral administration.

Estrogen Receptor α Mediates Doxorubicin Sensitivity in Breast Cancer Cells by Regulating E-Cadherin

Anthracyclines resistance is commonly seen in patients with estrogen receptor α (ERα) positive breast cancer. Epithelial-mesenchymal transition (EMT), which is characterized with the loss of epithelial cell polarity, cell adhesion and acquisition of new invasive property, is considered as one of the mechanisms of chemotherapy-induced drug resistance. In order to identify factors that associated with doxorubicin resistance, we performed in vitro and in vivo experiments using human and mouse breast cancer cell lines with different ERα status. Cell survival experiments revealed that ERα-positive cells (MCF-7 and MCF-7/ADR cell lines), were less sensitive to doxorubicin than ERα-negative (MDA-MB-231, MDA-MB-468) cells, and mouse mammary carcinoma cells (4T-1). The expression of E-cadherin reduced in low-invasive ERα-positive MCF-7 cells after treatment with doxorubicin, indicating epithelial mesenchymal transition. In contrast, the expression of E-cadherin was upregulated in high-invasive ERα-negative cells, showing mesenchymal-epithelial transition (MET). Moreover, it was found that the growth inhibition of 4T-1 cells by doxorubicin was positively correlated with the expression of E-cadherin. In a mouse breast cancer xenograft model, E-cadherin was overexpressed in the primary tumor tissues of the doxorubicin-treated mice. In ERα-positive MCF-7 cells, doxorubicin treatment upregulated the expression of EMT-related transcription factors Snail and Twist, that regulate the expression of E-cadherin. Following overexpression of ERα in ERα-negative cells (MDA-MB-231 and MDA-MB-468), doxorubicin enhanced the upregulation of Snail and Twist, decreased expression of E-cadherin, and decreased the sensitivity of cells to doxorubicin. In contrast, inhibition of ERα activity increased the sensitivity to doxorubicin in ERα-positive MCF-7 cells. These data suggest that the regulation of Snail and/or Twist varies depends on different ERα status. Therefore, doxorubicin combined with anti-estrogen receptor α therapy could improve the treatment efficacy of doxorubicin in ERα-positive breast cancer.

Metformin induces ferroptosis by targeting miR-324-3p/GPX4 axis in breast cancer.

Metformin is a widely prescribed hypoglycemic drug. Many studies have shown its anti-cancer properties. In the present study, we aimed to explore the effect of metformin on breast cancer and clarify the underlying mechanism. Our results showed that metformin induced ferroptosis in MDA-MB-231 cells through upregulating miR-324-3p expression. Overexpression of miR-324-3p inhibited cancer cell viability. miR-324-3p inhibitor promoted cell viability. Further studies showed that the effect of miR-324-3p was mediated by directly targeting glutathione peroxidase 4 (GPX4). miR-324-3p bound to the 3'-UTR of GPX4 and led to the downregulation of GPX4. In vivo studies showed that metformin induced ferroptosis by upregulating miR-324-3p in the xenograft model of breast cancer in mice. Our study suggested that metformin promotes ferroptosis of breast cancer by targeting the miR-324-3p/GPX4 axis. Metformin could act as a potential anti-cancer agent through the induction of ferroptosis.

Management of tumor growth and angiogenesis in triple-negative breast cancer by using redox nanoparticles

In cancer, angiogenesis is a critical phenomenon of nascent blood vessel development to facilitate the oxygen and nutrient supply prerequisite for tumor progression. Therefore, targeting tumors at the angiogenesis step may be significant to prevent their advanced progression and metastasis. Although angiogenesis inhibitors can limit the further growth of tumors, complete eradication of tumors may not be possible by monotherapy alone. Therefore, a therapeutic regimen targeting both tumor growth and its vasculature is essential. Because reactive oxygen species (ROS) are fundamental to both angiogenesis and tumor growth, the use of antioxidants may be an effective dual approach to inhibit tumors. We previously confirmed that our original antioxidant nitroxide radical-containing nanoparticles (RNPs) such as pH-sensitive RNPN, and pH-insensitive RNPO, effectively attenuates the tumorigenic and metastasis potentials of triple-negative breast cancer. In this study, we further investigated the efficacy of RNPs to limit the tumor progression by inhibiting the ROS-regulated cancer angiogenesis in a triple-negative breast cancer model. Here, we confirmed that RNPs significantly inhibited in vitro angiogenesis, attributed to the downregulation of the ROS-regulated angiogenesis inducer, vascular endothelial growth factor (VEGF) in the breast cancer cell line (MDA-MB231) and human umbilical vein endothelial cells (HUVEC), which was consistent with decreased cellular ROS. TEMPOL, a low-molecular-weight (LMW) control antioxidant, exhibited anti-angiogenic effects accompanied by cytotoxicity to the endothelial cells. In an in vivo xenograft model for breast cancer, RNPs exerted significant anti-tumor effect due to the decreased expression of tumor VEGF, which prevented accumulation of the endothelial cells. It should be noted that such efficacy of RNPs was obtained with negligible off-target effects. On the other hand, TEMPOL, because of its size, exerted anti-angiogenesis effect accompanied with injuries to the kidneys, which corroborated with previous reports. Our findings imply that RNPs are more potential antioxidants than their LMW counterparts, such as TEMPOL, for the management of breast cancers.

Hsp90 inhibitor-loaded IR780 micelles for mitochondria-targeted mild-temperature photothermal therapy in xenograft models of human breast cancer.

Mitochondria-targeted mild-temperature photothermal therapy (MT-PTT) is a promising strategy that can maximize anticancer effects and reduce adverse reactions. Here, a novel photosensitizer with mitochondrial targeting based on IR780 iodide and heat shock protein 90 inhibitor (BIIB021), which can passively accumulate in MCF-7cells and achieve effective MT-PTT effect is synthesized. The prepared PEG-IR780-BIIB021 nano-micelles possess considerable biocompatibility and biological stability, with an encapsulation efficiency of about 84% for BIIB021. They can selectively enrich in mitochondria, and release BIIB021 after NIR irradiation to reduce cell tolerance to heat, thereby reducing the mitochondrial membrane potential and rapidly affecting key intrinsic apoptotic factors (Cyt-C, Caspase-9, Bcl-2 and Bax) to achieve the effect of MT-PTT. It is believed that mitochondria-targeted MT-PTT generated by the PEG-IR780-BIIB021 nano-micelles is a promising therapeutic strategy in clinical practice.