Models Of Inflammatory Breast Cancer Research Articles

Abstract 5986: Significance of LIF/LIFR axis in the progression of inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare form of locally progressed breast cancer. Even though it is uncommon, IBC accounts for 7% of breast cancer deaths. There is a critical need for novel therapeutic targets to develop new therapeutics. IBC is more common in young women. Altered levels of growth factors, and cytokine signaling are suspected to play a role in the progression of IBC. Leukemia inhibitory factor (LIF) is the most pleiotropic member of the interleukin-6 family of cytokines. LIF and its receptor LIFR have been linked to the progression of many cancers including triple negative breast cancer. We recently developed a first-in-class inhibitor targeting LIFR named EC359. However, the role of the LIF/LIFR axis in IBC progression remains unknown. In this study, we examined the significance of LIFR and the effectiveness of LIFR inhibitor EC359 in treating IBC. Methods: The effect of LIFR inhibitor EC359 on cell survival, colony formation, and apoptosis was evaluated using three well-established IBC model cells (SUM149, SUM190PT, and KPL4). Mechanistic studies were conducted using Western blotting, CRISPR KO, reporter assays, and RT-qPCR. KPL4 cell-based xenografts were used to test the efficacy of EC359 in vivo. Results: Our results using Western blotting confirmed increased expression of LIF and LIFR in IBC cells compared to normal and ER+ breast cancer cells. Treatment with EC359 significantly decreased IBC cell viability, long term colony formation ability with an IC50 of ~10-20 nM. Similarly, knock out of LIFR significantly reduced the progression of IBC model cells in cell viability and colony formation assays. Further EC359 treatment promoted apoptosis of IBC cells. Mechanistic studies confirmed that EC359 treatment substantially reduced LIF/LIFR downstream signaling including STAT3, AKT, and mTOR signaling. We also tested the utility of EC359 in treating IBC in vivo using KPL4 xenografts. EC359 (5mg/kg/day) is highly efficacious in reducing IBC xenograft tumor growth in vivo. IHC analyses of tumors confirmed decreased cell proliferation as measured by Ki67 staining. Conclusion: Collectively, these studies demonstrated the therapeutic benefit of using EC359 to target the LIF/LIFR axis and opened the door for the development of new treatment approaches. Citation Format: Bianca Romo, Zenaida Fuentes, Lois Randolph, Bindu Santhamma, Hareesh Nair, Christoforos Thomas, Ratna Vadlamudi, Suryavathi Viswanadhapalli. Significance of LIF/LIFR axis in the progression of inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5986.

Abstract 2100: Novel LIPA targeted therapy for treating inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare but incredibly aggressive subtype of breast cancer (BC). IBC accounts for 2-4% of all occurrences of breast cancer and results in 7-10% of breast cancer-related deaths. IBC is only partially treatable with current therapies such as chemotherapy, surgery, and radiotherapy. Identification of novel therapeutic targets is urgently required. The main organelle for the synthesis, folding, and modification of proteins is called the endoplasmic reticulum (ER). Since elevated basal ER stress (ERS) is usually found in many cancers including IBC, our hypothesis was that the high basal ERS in IBC constitutes a serious vulnerability that can be targeted. Recently, we developed a small molecule ERX41 that promotes ER stress by blocking Lysosomal acid lipase A (LIPA) function in ER of cancer cells. The goal of the project is to test the utility of ERX-41 in treating IBC. Methods: To study the effect of ERX-41, we have used three well-established IBC model cells. We evaluated the efficacy of ERX-41 as a novel therapeutic for treating IBC using cell viability assays. The long-term effects of ERX-41 on IBC cell survival were evaluated using colony formation assays. Annexin V assays were used to measure apoptosis. Reporter assays, splicing assays, RT-qPCR, and Western blotting were used in mechanistic investigations. The effectiveness of ERX-41 was examined in vivo using KPL4 cell-based xenografts. Results: ERX-41 significantly decreased the viability of all three IBC model cells (SUM149, SUM190PT, and KPL4), with an IC50 of about 125 nM in MTT assay. Additionally, ERX-41 treatment significantly decreased IBC cells capacity to form colonies and promoted apoptosis. Specificity of the ERX41 mediated actions were confirmed using LIPA KO cells. Using RTqPCR based splicing assays, we demonstrated increased splicing of XBP1 as early as 6 hours after ERX41 treatment. Western analyses showed robust induction of stress markers (CHOP, PERK, ATF4) in IBC cells upon treatment with ERX-41. In KPL4 xenograft studies, ERX-41 showed significant reduction in the tumor volume. IHC analyses confirmed decreased proliferation marker and increased ER stress markers. Conclusions: Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of action and implicate ERX-41 binding to LIPA induces ER stress and promotes apoptosis of IBC cells. Citation Format: Zenaida Fuentes, Rahul Gopalam, Bianca A. Romo, Khaled Mohamed Nassar, Xue Yang, Behnam Ebrahimi, Paulina Ramirez, Chia-Yuan Chen, Scott Elmore, Harika Nagandla, Uday Pratap, Christoforos Thomas, Suryavathi Viswanadhapalli, Jung-Mo Ahn, Ganesh Raj, Ratna K. Vadlamudi. Novel LIPA targeted therapy for treating inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2100.

Abstract 6203: An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients

Abstract Background: Drug resistance is a major challenge in inflammatory breast cancer (IBC), a hyperproliferative subtype characterized by the presence of diffuse tumor cell clusters that show high level of heterogeneity. As the degree of tumor heterogeneity during cancer progression can cause evolution of cell states that can either compete or co-operate resulting in therapeutic failure, the study objective was to identify drug resistance networks that can serve as biomarkers or therapeutic targets. Methods: Integrated transcriptomic and proteomic profiling approach was conducted in a progression model of acquired drug resistance and its reversal comprised of a treatment-naïve patient-derived SUM149 cell line, along with two isogenic derivatives and representing drug-resistant (rSUM149) and resistance-reversal states (rrSUM149). Global gene expression analysis in IBC patients (n=87) recorded responses to neoadjuvant chemotherapy. Based on the data, targeted multikinase inhibitors were evaluated in viability and signaling assays. Results: We identified specific ribosomal proteins associated with resistance acquisition, which correlated with high levels of pERK, CDK1, XIAP, and SOD2. Conversely, the resistance reversal in rrSUM149 showed almost complete normalization to the profile of SUM149. However, VIPER analysis revealed proteins related to ribosomal processes (AGO2, Exportin 1, RPL5) that did not revert in rrSUM149, suggesting this pathway may have pleiotropic effects in governing drug resistance. Notably, genes linked to ribosomal processes were significantly enriched (P<0.001) among the overexpressed genes in IBC patients (n=87) who exhibited pCR to neoadjuvant chemotherapy. This is crucial as IBC tumors, including rSUM149, exhibit MAPK hyperactivation including in the presence of EGFR tyrosine kinase inhibitors. Treatment with Merestinib, a multikinase inhibitor, suppressed pERK, peIF4E, downstream targets like CDK1, and ribosomal protein IMP3 underscoring the impact on protein synthesis signaling crucial for countering translational dependence in cancer cells. Additionally, Merestinib synergized with EGFR-TKI (lapatinib) in increasing cell death in all three states. Conclusion: Overall, our study describes adaptive changes in response to both therapeutic stress and therapy break that have the potential to serve as biomarkers or extended for pharmacological interrogation toward precision medicine for this rare, understudied cancer. Furthermore, the ability of Merestinib, in clinical trials, to target translational dependence is attractive as cancer cells often appropriate these signaling pathways to gain tolerance to various stress stimuli. Support: NIH-R01CA264529; DoD Breast Cancer Breakthrough level 2 Award W81XWH2010153 Citation Format: Gayathri R. Devi, Pritha PAI, Matthew W. Foster, Dorababu S. Sannareddy, Seayoung Lee, Steven V. Laere. An altered profile of ribosomal proteins and ERK-eIF4E translational control in an inflammatory breast cancer model of acquired resistance and reversal is associated with pathological complete response in patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6203.

Targeting Estrogen Non‐Genomic Signaling in a Three‐Dimensional (3D) in VitroModel of Inflammatory Breast Cancer

Inflammatory Breast Cancer (IBC) is the most aggressive form of breast cancer with a low overall 5‐year survival rate of ~30‐60%. To this date the molecular signature associated with IBC has not been fully characterized, which lead to a lack of effective targeted therapeutics, especially for those patients that account for the 20‐40% of triple‐negative (TNBC) IBC cases. Several studies established that molecular signature of IBC shows important differences from the other breast cancer subtypes, such as overexpression of ErbB receptors, RhoC GTPase and Cox‐2, E‐cadherin aberrant expression, and loss of WISP3, among others. However, these molecular alterations do not explain completely the aggressive and rapid IBC progression. Recently, several studies have shown that estrogen can exert non‐genomic effect in triple‐negative IBC and other TNBCs, mediated by the expression of alternate estrogen receptors, including ERα36 and GPR30. Estrogen non‐genomic signaling refers to the rapid action of these alternate estrogen receptors that upon estrogen binding activates protein‐kinase cascades in the cytoplasm. We hypothesize that estrogen can elicit a specific non‐genomic signaling cascade that promotes the acquisition of aggressive oncogenic phenotypes in IBC and that targeting this pathway can be an effective anticancer therapy for IBC. To test this hypothesis, we used pharmacological inhibitors of the Epidermal Growth Factor Receptor (EGFR) alone and GPR30 alone or in combination. The first approach was to determine if inhibiting estrogen non‐genomic signaling by targeting one of the alternate receptors, GPR30, affects cell proliferation in a 3D colony formation assay using Matrigel. Preliminary observations showed that after G15 treatment (GPR30 inhibitor), there was a slight reduction in cell proliferation compared to E2, but when combined with IRESSA (EGFR inhibitor) there was a further decreased on proliferation when compared to IRESSA alone. Ongoing experiments include the effects of Icaritin (modulator of ERα36) and knockdown of ERα36 using siRNA experiments to test the effects in cell proliferation, migration and invasion. Also, we are in the process of determining the half‐maximal inhibitory concentration (IC50) of G15, IRESSA and IRESSA + G15 by performing a dose response curve (Alamar Blue cell viability assay) of SUM149 cell line, to observe if there is an additive effect when combined the pharmacological inhibitors of EGFR and GPR30. This study will provide the foundation for further characterization of the role estrogen non‐genomic signaling in the progression of IBC and to test its potential as a therapeutic target.

Abstract P5-08-13: NDRG1 expression is an independent prognostic factor in inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is a rare and highly aggressive form of breast cancer. Patients with IBC still have worse clinical outcomes compared to patients with non-IBC (40% versus 63% for 5 years overall survival, respectively) despite advances in breast cancer treatment. IBC remains a poorly characterized disease lacking specific therapeutic targets and prognostic biomarkers. Our group demonstrated that N-myc downstream regulated gene 1 (NDRG1) is crucial in promoting tumorigenesis and brain metastasis in mouse models of IBC. Our hypothesis is that NDRG1 is a prognostic marker associated with poor outcome in IBC patients. Methods: 64 IBC patients in a tissue microarray were evaluated by immunohistochemical staining with anti-NDRG1 primary antibody and NDRG1 levels were quantified. Using the median value, 32 patients were grouped as NDRG1-low (≤ median), and 32 as NDRG1-high (>median). Survival data were compared by Kaplan–Meier curves and log-rank test. Results: The average age of patients was 50 years. Sixty-two percent of patients had estrogen receptor (ER)-negative tumors, 83% were stage III, 80% high grade, and 67% of these patients received adjuvant radiation. The median follow-up time for the patients studied was 11.7 years, and the median overall survival (OS) time was 3.7 years. On univariate analysis, NDRG1 expression, tumor grade, disease stage, ER status, and adjuvant radiation therapy were associated with OS and disease specific survival (DSS). Patients with NDRG1-low tumors experienced better actuarial 10-year OS (p=0.0129) and DSS (p=0.0074), and showed significant higher 10-year OS and DSS rates than patients with NDRG1-high(OS, 45% vs. 19%, p=0.0278; DSS, 52% vs. 22%, p=0.0139). The median OS and DSS times were shorter for NDRG1-high patients (OS, 2.5 years; DSS, 3.1 years) than for NDRG1-low patients (OS, 5.9 years; DSS, 10.7 years). Multivariable analysis, NDRG1 was an independent predictor of OS (hazard ratio [HR]=2.449, p=0.0274) and DSS (HR=2.727, p=0.0039). ER status, disease stage and adjuvant radiation were also independent predictor for both OS and DSS. Furthermore, we observed that NDRG1-high expressing ER-negative tumors exhibit worse outcome in IBC patients (OS, p=0.0003; DSS, p=0.0003). NDRG1-high expression correlated with worse outcome in patients that received adjuvant radiation treatment (OS, p=0.0088; DSS, p=0.0093), and those with stage III (OS, p=0.0450; DSS, p=0.0239). Conclusions: Our findings demonstrated that NDRG1 is positively correlated with aggressive tumor characteristics in IBC and that it is an independent prognostic factor for OS and DSS in IBC patients, suggesting that targeting NDRG1 may provide a novel therapeutic strategy to improve outcomes for patients with IBC. Our data further suggests that NDRG1 warrants further investigation in radiation response. Citation Format: Emilly Schlee Villodre, Yun Gong, Xiaoding Hu, Lei Huo, Esther C Yoon, Naoto T Ueno, Wendy A Woodward, Debu Tripathy, Juhee Song, Bisrat G Debeb. NDRG1 expression is an independent prognostic factor in inflammatory breast cancer [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 P5-08-13.

Comparative transcriptional analyses of preclinical models and patient samples reveal MYC and RELA driven expression patterns that define the molecular landscape of IBC

Inflammatory breast cancer (IBC) is an aggressive disease for which the spectrum of preclinical models was rather limited in the past. More recently, novel cell lines and xenografts have been developed. This study evaluates the transcriptome of an extended series of IBC preclinical models and performed a comparative analysis with patient samples to determine the extent to which the current models recapitulate the molecular characteristics of IBC observed clinically. We demonstrate that the IBC preclinical models are exclusively estrogen receptor (ER)-negative and of the basal-like subtype, which reflects to some extent the predominance of these subtypes in patient samples. The IBC-specific 79-signature we previously reported was retrained and discriminated between IBC and non-IBC preclinical models, but with a relatively high rate of false positive predictions. Further analyses of gene expression profiles revealed important roles for cell proliferation, MYC transcriptional activity, and TNFɑ/NFκB in the biology of IBC. Patterns of MYC expression and transcriptional activity were further explored in patient samples, which revealed interactions with ESR1 expression that are contrasting in IBC and nIBC and notable given the comparatively poor outcomes of ER+ IBC. Our analyses also suggest important roles for NMYC, MXD3, MAX, and MLX in shaping MYC signaling in IBC. Overall, we demonstrate that the IBC preclinical models can be used to unravel cancer cell intrinsic molecular features, and thus constitute valuable research tools. Nevertheless, the current lack of ER-positive IBC models remains a major hurdle, particularly since interactions with the ER pathway appear to be relevant for IBC.

High endogenous CCL2 expression promotes the aggressive phenotype of human inflammatory breast cancer

Inflammatory Breast Cancer (IBC) is a highly aggressive malignancy with distinct clinical and histopathological features whose molecular basis is unresolved. Here we describe a human IBC cell line, A3250, that recapitulates key IBC features in a mouse xenograft model, including skin erythema, diffuse tumor growth, dermal lymphatic invasion, and extensive metastases. A3250 cells express very high levels of the CCL2 chemokine and induce tumors enriched in macrophages. CCL2 knockdown leads to a striking reduction in macrophage densities, tumor proliferation, skin erythema, and metastasis. These results establish IBC-derived CCL2 as a key factor driving macrophage expansion, and indirectly tumor growth, with transcriptomic analysis demonstrating the activation of multiple inflammatory pathways. Finally, primary human IBCs exhibit macrophage infiltration and an enriched macrophage RNA signature. Thus, this human IBC model provides insight into the distinctive biology of IBC, and highlights potential therapeutic approaches to this deadly disease.

Accumulation of amyloid beta (Aβ) and amyloid precursor protein (APP) in tumors formed by a mouse xenograft model of inflammatory breast cancer.

Accumulation of amyloid in breast cancer is a well‐known phenomenon, but only immunoglobulin light‐chain amyloidosis (AL) or transthyretin (TTR) amyloid had been detected in human breast tumor samples previously. We recently reported that another amyloidogenic peptide, amyloid beta (Aβ), is present in an aggregated form in animal and human high‐grade gliomas and suggested that it originates systemically from the blood, possibly generated by platelets. To study whether breast cancers are also associated with these Aβ peptides and in what form, we used a nude mouse model inoculated with triple‐negative inflammatory breast cancer cell (SUM‐149) xenografts, which develop noticeable tumors. Immunostaining with two types of specific antibodies for Aβ identified the clear presence of Aβ peptides associated with (a) carcinoma cells and (b) extracellular aggregated amyloid (also revealed by Congo red and thioflavin S staining). Aβ peptides, in both cells and in aggregated amyloid, were distributed in clear gradients, with maximum levels near blood vessels. We detected significant presence of amyloid precursor protein (APP) in the walls of blood vessels of tumor samples, as well as in carcinoma cells. Finally, we used ELISA to confirm the presence of elevated levels of mouse‐generated Aβ40 in tumors. We conclude that Aβ in inflammatory breast cancer tumors, at least in a mouse model, is always present and is concentrated near blood vessels. We also discuss here the possible pathways of Aβ accumulation in tumors and whether this phenomenon could represent the specific signature of high‐grade cancers.

Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion.

Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer characterized by rapid onset and high risk of metastasis and poor clinical outcomes. The biological basis for the aggressiveness of IBC is still not well understood and no IBC‐specific targeted therapies exist. In this study, we report that lipocalin 2 (LCN2), a small secreted glycoprotein belonging to the lipocalin superfamily, is expressed at significantly higher levels in IBC vs non‐IBC tumors, independently of molecular subtype. LCN2 levels were also significantly higher in IBC cell lines and in their culture media than in non‐IBC cell lines. High expression was associated with poor‐prognosis features and shorter overall survival in IBC patients. Depletion of LCN2 in IBC cell lines reduced colony formation, migration, and cancer stem cell populations in vitro and inhibited tumor growth, skin invasion, and brain metastasis in mouse models of IBC. Analysis of our proteomics data showed reduced expression of proteins involved in cell cycle and DNA repair in LCN2‐silenced IBC cells. Our findings support that LCN2 promotes IBC tumor aggressiveness and offer a new potential therapeutic target for IBC.

NDRG1 Expression Is an Independent Prognostic Factor in Inflammatory Breast Cancer.

Simple SummaryInflammatory breast cancer (IBC) is a rare and aggressive variant of breast cancer that is responsible for a significant number of breast cancer-related deaths. Herein, we describe how the expression of a specific protein named N-myc downstream-regulated gene 1 (NDRG1), commonly described as a gene that prevents the spread of cancer cells to distant organs, may have a paradoxical role in cancer progression in IBC. We found that the level of expression of NDRG1 in tumor tissues predicts the survival outcome of patients with IBC. We also observed that NDRG1, together with other important prognostic factors such as estrogen receptor status and stage, could be used to further analyze prognostic outcome or treatment response of patients.NDRG1 is widely described as a metastasis suppressor in breast cancer. However, we found that NDRG1 is critical in promoting tumorigenesis and brain metastasis in mouse models of inflammatory breast cancer (IBC), a rare but highly aggressive form of breast cancer. We hypothesized that NDRG1 is a prognostic marker associated with poor outcome in patients with IBC. NDRG1 levels in tissue microarrays from 64 IBC patients were evaluated by immunohistochemical staining with NDRG1 (32 NDRG1-low (≤median), 32 NDRG1-high (>median)). Overall and disease-free survival (OS and DSS) were analyzed with Kaplan–Meier curves and log-rank test. Univariate analysis showed NDRG1 expression, tumor grade, disease stage, estrogen receptor (ER) status, and receipt of adjuvant radiation to be associated with OS and DSS. NDRG1-high patients had poorer 10-year OS and DSS than NDRG1-low patients (OS, 19% vs. 45%, p = 0.0278; DSS, 22% vs. 52%, p = 0.0139). On multivariable analysis, NDRG1 independently predicted OS (hazard ratio (HR) = 2.034, p = 0.0274) and DSS (HR = 2.287, p = 0.0174). NDRG1-high ER-negative tumors had worse outcomes OS, p = 0.0003; DSS, p = 0.0003; and NDRG1-high tumors that received adjuvant radiation treatment had poor outcomes (OS, p = 0.0088; DSS, p = 0.0093). NDRG1 was a significant independent prognostic factor for OS and DSS in IBC patients. Targeting NDRG1 may represent a novel strategy for improving clinical outcomes for patients with IBC.

Targeting estrogen non‐genomic signaling in a three‐dimensional (3D) in vitro model of inflammatory breast cancer

Inflammatory Breast Cancer (IBC) is the most aggressive form of breast cancer with a low overall 5‐year survival rate of ~30–60%. To this date the molecular signature associated with IBC is not completely characterized, which lead to a lack of effective targeted therapeutics, especially for those patients that account for the 20–40% of triple‐negative (TNBC) IBC cases. Several studies established that molecular signature of IBC shows important differences from the other breast cancer subtypes, such as overexpression of ErbB receptors, RhoC GTPase and Cox‐2, E‐cadherin aberrant expression, and loss of WISP3 among others. However, these molecular alterations do not explain completely the aggressive and rapid IBC phenotype. Recently, several studies have shown that estrogen can exert non‐genomic effects in triple‐negative IBC and other TNBCs, mediated by the expression of alternate estrogen receptors, including ERα36 and GPR30. Estrogen non‐genomic signaling refers to the rapid action of these alternate estrogen receptors that upon estrogen binding activates protein‐kinase cascades in the cytoplasm. We hypothesize that estrogen can elicit a specific non‐genomic signaling cascade that promotes the acquisition of aggressive oncogenic phenotypes in IBC and that targeting this pathway can be an effective anticancer therapy for IBC. To test this hypothesis, we are going to use pharmacological inhibition of ERα36 and GPR30 using antagonists against these receptors alone or in combination with EGFR inhibition. In parallel, we are going to use siRNA to down‐regulate the expression of these receptors. The first approach is to determine if inhibiting estrogen nongenomic signaling by targeting the alternate receptors affect cell proliferation in a 3D colony formation assay using Matrigel. Preliminary observations showed that cells treated with E2 develop larger colonies (increase proliferation) when compared to vehicle control. Inhibiting GPR30 alone have a slight reduction in cell proliferation compared to E2, but when combined with an EGFR inhibitor (Gefitinib) showed a synergistic effect by decreasing proliferation further that by Gefitinib alone. Ongoing experiments include the effects of Icaritin (modulator of ERα36) and the siRNA experiments to test the effects in cell proliferation, migration and invasion. This study will provide the foundation for further characterization of the role estrogen non‐genomic signaling in the progression of IBC and to test its potential as a therapeutic target.Support or Funding InformationSupport by RISE Program Grant number: 5R25GM061151‐18

Abstract P6-06-08: Transcriptome analysis of patient derived xenograft mouse models of inflammatory breast cancer to gain insights into the contribution of tumor microenvironment

Abstract Purpose: Inflammatory breast cancer (IBC) is an aggressive variant of breast cancer characterized by visible skin symptoms on the breast at the time of presentation. We demonstrated mesenchymal stem cells (MSC) promote skin symptoms in the SUM149 xenograft model. Here, we extend this finding to IBC patient-derived xenograft (PDX) lines and performed RNA-sequencing analysis of tumors from IBC patient-derived xenograft (PDX) lines by presence of skin symptoms. We hypothesize host MSC variation may explain sporadic skin symptoms in IBC PDX tumors across generations. Methods: MSC were co-injected at the time of tumor initiation in PDX tumors (IBC-3, 5, 6, and 7) and skin symptoms assessed visually and at the time of gross resection. A priori analysis plan was to group PDX by treatment group irrespective of PDX line. IBC PDX models spontaneously but inconsistently develop skin symptoms after multiple passages. RNA sequencing was performed on IBC-PDX tumors (n=33) and non-IBC PDX tumors (n=12) across generations from IBC PDX lines 5, 6, and 7 and non-IBC PDX lines 2, 3, and 4. Four samples were discarded due to poor quality. TSNE was applied using the top differential expressed genes. Xenome analysis was performed to examine host stromal expression by skin involvement. Samples from PDX lines 5 were compared with lines 6 and 7 and the top differentially expressed genes were compared with the Molecular Signatures Databases (MSigDB). CIBERSORT was performed to deconvolute cell type composition. Results: Co-injecting MSC with PDX transplant increased skin symptoms (57% of PDX animals (cohort from IBC5, IBC6, IBC7 and non-IBC4) with MSC vs. 0% without (cohort from IBC5, IBC6, IBC7, non-IBC2 and non-IBC4) (P = 0.0007). TSNE analysis of IBC PDX split samples into three subgroups distinguished by PDX lines and skin invasion. Xenome was used to split reads from human or mouse: samples show high percentage of reads coming from human (mean±SD, 97.44%± 8.98%). Seventy-four genes show FDR ≤ 0.2 between PDX line 5 and line 6 and comparing these top 74 genes with MSigDb for interaction revealed pathways such as GPCR, signal transduction, and sensory perception. CIBERSORT revealed no enrichment for MSC in IBC PDX with skin invasion, however significant differences in CD8 expression in these immunosuppressed PDX models casts doubt on the robustness of this finding. Conclusions: Exogenous MSC enhance skin symptoms in a cohort of IBC and non-IBC PDX. Skin symptoms segregate IBC PDX tumors by expression suggesting specific biology drives this presentation. However, we are unable to demonstrate evidence for spontaneous MSC signaling in these models. Future work is warranted to link top skin symptom related signaling to stromal signals and unravel the unexpected CD8 signaling identified in these models. Citation Format: Omar Rahal, Jie Yang, Li Li, Richard Larson, Steven Van Laere, Naoto Ueno, Erik Sulman, Wendy Woodward. Transcriptome analysis of patient derived xenograft mouse models of inflammatory breast cancer to gain insights into the contribution of tumor microenvironment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-06-08.

PARP1 Inhibition Radiosensitizes Models of Inflammatory Breast Cancer to Ionizing Radiation.

Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC), with local control rates of 80% or less at 5 years. Given the unsatisfactory outcomes for these patients, there is a clear need for intensification of local therapy, including radiation. Inhibition of the DNA repair protein PARP1 has had little efficacy as a single agent in breast cancer outside of studies restricted to patients with BRCA mutations; however, PARP1 inhibition (PARPi) may lead to the radiosensitization of aggressive tumor types. Thus, this study investigates inhibition of PARP1 as a novel and promising radiosensitization strategy in IBC. In multiple existing IBC models (SUM-149, SUM-190, MDA-IBC-3), PARPi (AZD2281-olaparib and ABT-888-veliparib) had limited single-agent efficacy (IC50 > 10 μmol/L) in proliferation assays. Despite limited single-agent efficacy, submicromolar concentrations of AZD2281 in combination with RT led to significant radiosensitization (rER 1.12-1.76). This effect was partially dependent on BRCA1 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA breaks as measured by multiple assays. Using a SUM-190 xenograft model in vivo, the combination of PARPi and RT significantly delays tumor doubling and tripling times compared with PARPi or RT alone with limited toxicity. This study demonstrates that PARPi improves the effectiveness of radiotherapy in IBC models and provides the preclinical rationale for the opening phase II randomized trial of RT ± PARPi in women with IBC (SWOG 1706, NCT03598257).

Abstract 3924: PARP inhibition as a radiosensitizing strategy to improve locoregional control in inflammatory breast cancer

Abstract Purpose: Sustained locoregional control of disease is a significant issue in patients with inflammatory breast cancer (IBC) with local control rates <70% at 5 years in these patients. Given the unsatisfactory outcomes, there is a clear need for intensification of local therapy, including radiation, for these patients. Inhibition of poly(adenosine diphosphate-ribose) polymerase 1 (PARP1), a DNA repair protein, represents a novel and promising strategy for radiosensitizing aggressive tumors. The purpose of this study was to investigate radiosensitization by PARP1 inhibition in IBC and to determine the mechanism of radiosensitization. Methods: Pharmacologic PARP1 inhibitors, olaparib and veliparib, were used at various concentrations (10-5 to 10-12 M). Proliferation assays were used to determine the IC50s of these inhibitors in three independent IBC cell lines (SUM 149, SUM 190, MDA-IBC-3). Clonogenic survival assays were used to determine the radiosensitization in cell lines after pharmacologic PARP inhibition (PARPi). DNA damage was quantified using γH2AX staining in IBC cell lines. Transcriptomic, protein and signaling pathway changes were measured using RNA-Seq and RPPA, respectively. In vivo tumor growth was also measured in CB17-SCID mice with varying control and treatment groups (16-20 tumors/group) that included radiation (RT) alone, olaparib alone, and RT+olaparib. Results: PARPi via olaparib and veliparib had limited single agent efficacy in all three independent IBC models with IC50 values >10 µM. Despite limited single agent efficacy, sub micromolar concentrations of PARPi with RT led to significant radiosensitization with radiation enhancement ratios of 1.20-1.35 in the same three models (500 nM in SUM 190, 500 nM in MDA-IBC-3, 2 nM in SUM 149). This effect was partially dependent on BRCA 1/2 mutational status. Radiosensitization was due, at least in part, to delayed resolution of double strand DNA (dsDNA) breaks at 12, 16, and 24 hours in all models. Additionally, this effect is mediated, at least in part, by a non-homologous end joining (NHEJ) mechanism and was not dependent on homologous recombination (HR). RNA-Seq and RPPA protein pathway data identify key pathways that regulate the radiosensitization in these IBC models. Additionally, xenograft studies show significant differences in tumor doubling and tripling times between treated and untreated tumors. Conclusion: Our study demonstrates that the PARP1 inhibition improves the therapeutic index of radiotherapy in IBC cell lines. Combined PARPi with RT leads to unresolved dsDNA breaks and decreased clonogen survival. These studies provide the rationale for a phase II randomized trial of RT +/- PARPi (olaparib) in women with IBC that has recently opened for accrual (SWOG 1706 - NCT03598257). This work was supported by a grant from Komen for the Cure and NIH T-32-GM007315. Citation Format: Anna Michmerhuizen, Andrea Pesch, Leah Moubadder, Meleah Cameron, Amanda Zhang, Nicole Hirsh, Meilan Liu, Kari Wilder-Romans, Lori J. Pierce, Reshma Jagsi, Corey Speers. PARP inhibition as a radiosensitizing strategy to improve locoregional control in inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3924.

Synthesis, structure-activity relationship and in vitro pharmacodynamics of A-ring modified caged xanthones in a preclinical model of inflammatory breast cancer

Inflammatory breast cancer (IBC) is a highly metastatic, lethal form of breast cancer that lacks targeted therapeutic strategies. Inspired by the promising cytotoxicity of gambogic acid and related caged xanthones in spheroidsMARY-X, an in vitro preclinical IBC model, we constructed a library of synthetic analogs and performed structure-activity relationship studies. The studies revealed that functionalizing the A-ring of the caged xanthone framework can significantly affect potency. Specifically, introduction of hydroxyl or fluorine groups at discrete positions of the A-ring leads to enhanced cytotoxicity at submicromolar concentrations. These compounds induce complete dissolution of spheroidsMARY-X with subsequent apoptosis of both the peripherally- and centrally-located cells, proliferative and quiescent-prone (e.g. hypoxic), respectively. These results highlight the structural flexibility and pharmacological potential of the caged xanthone motif for the design of IBC-targeting therapeutics.

Identification of Biologically Active Ganoderma lucidum Compounds and Synthesis of Improved Derivatives That Confer Anti-cancer Activities in vitro.

We previously reported that Ganoderma lucidum extract (GLE) demonstrate significant anti-cancer activity against triple negative inflammatory breast cancer models. Herein, we aimed to elucidate the bioactive compounds of GLE responsible for this anti-cancer activity. We performed NMR, X-ray crystallography and analog derivatization as well as anti-cancer activity studies to elucidate and test the compounds. We report the structures of the seven most abundant GLE compounds and their selective efficacy against triple negative (TNBC) and inflammatory breast cancers (IBC) and other human cancer cell types (solid and blood malignancies) to illustrate their potential as anti-cancer agents. Three of the seven compounds (ergosterol, 5,6-dehydroergosterol and ergosterol peroxide) exhibited significant in vitro anti-cancer activities, while we report for the first time the structure elucidation of 5,6-dehydroergosterol from Ganoderma lucidum. We also show for the first time in TNBC/IBC cells that ergosterol peroxide (EP) displays anti-proliferative effects through G1 phase cell cycle arrest, apoptosis induction via caspase 3/7 activation, and PARP cleavage. EP decreased migratory and invasive effects of cancer cells while inhibiting the expression of total AKT1, AKT2, BCL-XL, Cyclin D1 and c-Myc in the tested IBC cells. Our investigation also indicates that these compounds induce reactive oxygen species, compromising cell fate. Furthermore, we generated a superior derivative, ergosterol peroxide sulfonamide, with improved potency in IBC cells and ample therapeutic index (TI > 10) compared to normal cells. The combined studies indicate that EP from Ganoderma lucidum extract is a promising molecular scaffold for further exploration as an anti-cancer agent.

Abstract P5-17-01: Patient-derived organoid models of inflammatory breast cancer

Abstract Background: Inflammatory Breast Cancer (IBC) is an aggressive subtype of breast cancer that is associated with an increased likelihood of early metastasis and a poor prognosis, accounting for approximately 10% of all breast cancer mortality. Currently, there is limited availability of cell line and patient-derived xenograft (PDX) models of IBC. As a result, there is an urgent need for additional model systems to facilitate our understanding of this disease. Organoids have been used to grow breast cancer cells with high efficiency, resulting in long-term cultures with genetic and phenotypic stability; however, the extent to which IBC can be modeled using this system is unknown. We undertook an evaluation of organoid culture technology for growing IBC patient samples, with the goal of generating a model of IBC that more faithfully recapitulates the key features of the disease. Methods: Tumor samples (taken from lumpectomy, mastectomy, PDX, or core biopsy samples), were digested to the organoid level using collagenase, and were grown in three dimensional cultures using a basement membrane extract and a fully-defined organoid medium, as described (Sachs et al. Cell 2018). First, we surveyed non-IBC metastatic breast cancer samples to determine the efficiency of growth of multiple breast cancer subtypes under these conditions. Then, we assessed the efficiency of growth of IBC samples taken after treatment, at the time of mastectomy. Co-culture experiments in which IBC organoids were grown in the presence of human endothelial cells were performed in three dimensional culture using a mixture of type I collagen and basement membrane extract. Results: Our success rate for establishing organoid cultures from breast cancer samples in general was 75% (33 out of 44 samples), and for triple-negative breast cancers, the success rate for establishment of organoid cultures was 100% (14 out of 14 samples). Expansion of ER+ tumor organoids (16 of 26 samples) was primarily restricted by a slow growth rate in culture, as compared to triple-negative tumor organoids. We were able to establish 12 breast cancer organoids from core biopsies of multiple metastatic sites, including brain, liver, bone, breast, and skin. We subsequently were able to establish organoid cultures from five out of five IBC tumor samples. Our IBC organoids preserved histopathologic features of the original tumor, and expressed key markers, such as E-cadherin, that are known to be associated with IBC. In addition, our IBC organoids interacted with human endothelial cells under co-culture conditions designed to model lymphovascular invasion, an essential feature of IBC. Conclusion: We have shown that organoid cultures can be established to propagate IBC tumors with high efficiency, preserve key pathologic features of IBC in vitro, and model interactions between tumor cells and the microenvironment. Thus, organoids are useful complements to existing IBC models, and can be used to identify potential therapeutic vulnerabilities associated with this particularly aggressive breast cancer subtype. Citation Format: Rosenbluth JM, Zervantonakis I, Boedicker M, Wagle N, Dillon D, Nakhlis F, Brugge JS, Overmoyer B. Patient-derived organoid models of inflammatory breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-01.

Abstract P2-01-03: Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal form of primary breast cancer and accounts for a significant 10 % of breast cancer deaths in the USA owing to its aggressive proliferation and metastasis, and a lack of effective therapeutic options. Unraveling the underlying mechanisms of growth and metastasis of this aggressive disease could lead to effective therapeutic strategies for an improved outcome in IBC patients. We recently generated in vitro and in vivo IBC models for brain metastasis studies [Debeb et al. JNCI, 2016] and observed an upregulation of Lipocalin 2 (LCN2), a small, secreted iron-trafficking protein which plays a significant role in immune and inflammatory responses and the promotion of malignant progression. The purpose of this study was to investigate the function of LCN2 in IBC tumorigenesis and metastasis. Methods: Stable knockdown (KD) of LCN2 in IBC cell lines was achieved with lentiviral vectors. Proteomic and gene expression profiling were performed using RPPA and Affymetrix Clariom D microarray. For in vivo studies, control and LCN2 KD IBC cells were transplanted into the cleared mammary fat pad of SCID/Beige mice. Tumor-skin involvement was assessed visually during primary tumor growth and tumor excision. LCN2 gene expression levels in clinical samples were analyzed from the IBC Consortium as well as public data sets. LCN2 serum levels in IBC patients were measured using ELISA and were correlated with clinicopathological variables and outcome data. Results: LCN2 gene expression is higher in IBC versus non-IBC patients (p=0.00036), independently of the molecular subtypes, and higher in more aggressive (TNBC and HER2+) than hormone receptor-positive subtypes (p<0.00001). LCN2 expression in patient tissues is correlated with reduced overall survival (p<0.00001) and metastasis-free survival (p=0.04) in non-IBC; however, LCN2 was not associated with overall survival in IBC patient serum samples. LCN2 expression was also significantly higher in IBC cell lines, in their culture media, and in brain metastasis sublines compared to non-IBC cell lines (p=0.004). In IBC cell lines, LCN2 KD reduced proliferation, colony formation, migration, and cancer stem cell properties. In vivo silencing of LCN2 in SUM149 cells inhibited primary tumor growth (p=0.001)and resulted in a well-differentiated tumor histology. Additionally, SUM149 LCN2 KD significantly reduced skin invasion/recurrence (LCN2 control vs LCN2 KD: 88 % vs 25 %, p=0.01) suggesting LCN2 is a mediator of tumorigenesis. Analysis of proteomics data showed changes in major signaling pathways including PI3K-Akt signaling and EGF/EGFR signaling pathways. Mechanistically, LCN2 depletion in SUM149 abrogated EGF-induced EGFR phosphorylation and ERK activation. Conclusions: Our findings suggest that LCN2 may drive IBC tumor progression and skin invasion/recurrence potentially via the EGFR signaling pathway.Future studies will determine the role of LCN2 in metastasis and pinpoint the detailed mechanisms of LCN2-mediated IBC tumorigenesis and recurrence. Citation Format: Villodre ES, Larson R, Hu X, Stecklein SR, Gomez K, Finetti P, Krishnamurthy S, Ivan C, Su X, Ueno NT, Van Laere S, Bertucci F, Tripathy D, Vivas-Mejía P, A Woodward W, Debeb BG. Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-03.

Abstract 2397: Intracellular cholesterol regulates the DNA damage response in inflammatory breast cancer

Abstract Purpose: Inflammatory breast cancer (IBC) is a highly aggressive form of breast cancer, and patients with IBC remain at high risk of locoregional recurrence after radiotherapy. We previously demonstrated that depleting intracellular cholesterol induces radiosensitivity in IBC cells in vitro, and that IBC patients with high levels of high-density lipoprotein (HDL) and those taking a statin, which inhibits de novo cholesterol biosynthesis, have improved locoregional control after radiotherapy. These results suggest that targeting cholesterol metabolism may improve the radiotherapeutic management of IBC. Here, we aimed to understand the molecular mechanism(s) linking cholesterol metabolism and radiation-induced DNA damage and DNA repair and to evaluate statin pharmacotherapy as a DNA repair inhibitor and radiotherapeutic adjunct in an in vivo pre-clinical model of IBC. Methods: KPL4, SUM149, SUM190, and IBC3 IBC cell lines were used to examine the effect(s) of simvastatin and human serum lipoproteins on radiation-induced DNA damage induction, DNA damage signaling, DNA damage-associated G1, intra-S, and G2/M cell cycle checkpoint activation, and DNA double strand break (DSB) repair by homologous recombination (HR) and non-homologous end joining (NHEJ). Results: We have generated multiple IBC cells lines with an integrated Traffic Light Reporter (TLR) that is capable of measuring HR and NHEJ, and demonstrate that depletion of intracellular cholesterol abrogates HR-mediated repair of DNA DSBs. We also show that intracellular cholesterol has pleiotropic effects on cell cycle distribution and DNA damage-associated cell cycle checkpoints. We also report a novel polycistronic dual bioluminescence reporter system that can robustly visualize and quantitate DNA DSB induction and repair in vitro and in vivo. Conclusions: Cholesterol is an important determinant of intrinsic radiosensitivity in IBC, and appears to regulate repair of radiation-induced DNA DSBs. Targeting cholesterol metabolism is a potential strategy to overcome IBC radioresistance and improve the prognosis for this aggressive disease. Citation Format: Shane R. Stecklein, Adam R. Wolfe, Bisrat G. Debeb, Richard A. Larson, Wendy A. Woodward. Intracellular cholesterol regulates the DNA damage response in inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2397.

Abstract B47: Efficacy of GLI antagonists in triple-negative inflammatory breast cancer 2D and 3D models

Abstract Our objective was to investigate the differential effects of specific GLI1 antagonists in triple-negative inflammatory breast cancer 2D and 3D cell culture systems. Inflammatory breast cancer (IBC) accounts for 15% of all breast cancer deaths. IBC is a unique form of locally advanced breast cancer, an aggressive and highly invasive breast cancer having one of the worst clinical outcomes among breast cancers. African-American, American-Indian, and Arab-American women are disproportionately affected by IBC (1-3). Compared to non-IBC, IBC has a higher incidence of the triple-negative (TN) phenotype for which there are few treatment options. There are currently limited strategies for targeting IBC; thus, IBC patients continue to have worse survival outcomes than non-IBC breast cancer patients. GLI1 (glioma-associated oncogene homolog 1), the terminal effector of the hedgehog (Hh) pathway, has emerged as a potential therapeutic target for TN breast cancers including IBC (4). There is overwhelming evidence for GLI1 activation across many tumor types, including aggressive breast cancers, and its importance for tumor progression. Nuclear expression of GLI1 has been shown to be a strong predictor of poor prognosis in pancreatic cancer and brain gliomas and correlates with aggressiveness in basal-like breast cancer. In our studies, we utilized various automated phenotypic and functional assays (5) to examine the efficacy of GLI antagonists with varying mechanism of action for effects on IBC cell proliferation and growth in 2D and 3D cell culture systems. We are also assessing by high-throughput transcriptome sequencing (RNA-seq) gene expression differences in IBC cell lines after treatment with GLI antagonists. In both our 2D and 3D cell culture systems, TN-IBC cell lines were highly sensitive to two GLI antagonists, JK184 and GANT61. JK184 and GANT61 showed significant but differential effects on TN-IBC cell proliferation, colony formation, cell motility, and spheroid formation. This study was supported in part by Department of Defense award W81WXH-13-1-0141, NIH award P20CA202924, Komen Graduate Training in Disparities Research award GTDR16377604, Developmental Funds from Duke School of Medicine and Duke Cancer Institute (as part of the P30CA014236 and Cancer and Environment Program supported by Mr and Mrs Fred and Alice Stanback P3917733).