Model Of Breast Cancer Progression Research Articles

TGFβ1-TNFα regulated secretion of neutrophil chemokines is independent of epithelial-mesenchymal transitions in breast tumor cells.

Neutrophils have tumor-promoting roles in breast cancer and are detected in higher numbers in aggressive breast tumors. How aggressive breast tumors recruit neutrophils remains undefined. Here, we investigated the roles of TGF-β1 and TNF-α in the regulation of neutrophil recruitment by breast cancer cells. TGF-β1 and TNF-α are pro-inflammatory factors upregulated in breast tumors and induce epithelial to mesenchymal transitions (EMT), a process linked to cancer cell aggressiveness. We report that, as expected, dual treatment with TGF-β1 and TNF-α induces EMT signatures in premalignant M2 cells, which are part of the MCF10A breast cancer progression model. Conditioned media (CM) harvested from M2 cells treated with TGF-β1/TNF-α gives rise to amplified neutrophil chemotaxis compared to CM from control M2 cells. This response correlates with higher levels of the neutrophil chemokines CXCL1, CXCL2, and CXCL8 and is significantly attenuated in the presence of a CXCL8-neutralizing antibody. Furthermore, we found that secretion of CXCL1 and CXCL8 from treated M2 cells depends on p38MAPK activity. By combining gene editing, immunological and biochemical approaches, we show that the regulation of neutrophil recruitment and EMT signatures are not mechanistically linked in treated M2 cells. Finally, analysis of publicly available cancer cell line transcriptomic databases revealed a significant correlation between CXCL8 and TGF-β1/TNF-α-regulated or effector genes in breast cancer. Together, our findings establish a novel role for the TGF-β1/TNF-α/p38 MAPK signaling axis in regulating neutrophil recruitment in breast cancer, independent of TGF-β1/TNF-α regulated EMT.

Loss of multi-level 3D genome organization during breast cancer progression.

Breast cancer entails intricate alterations in genome organization and expression. However, how three-dimensional (3D) chromatin structure changes in the progression from a normal to a breast cancer malignant state remains unknown. To address this, we conducted an analysis combining Hi-C data with lamina-associated domains (LADs), epigenomic marks, and gene expression in an in vitro model of breast cancer progression. Our results reveal that while the fundamental properties of topologically associating domains (TADs) are overall maintained, significant changes occur in the organization of compartments and subcompartments. These changes are closely correlated with alterations in the expression of oncogenic genes. We also observe a restructuring of TAD-TAD interactions, coinciding with a loss of spatial compartmentalization and radial positioning of the 3D genome. Notably, we identify a previously unrecognized interchromosomal insertion event, wherein a locus on chromosome 8 housing the MYC oncogene is inserted into a highly active subcompartment on chromosome 10. This insertion is accompanied by the formation of de novo enhancer contacts and activation of MYC , illustrating how structural genomic variants can alter the 3D genome to drive oncogenic states. In summary, our findings provide evidence for the loss of genome organization at multiple scales during breast cancer progression revealing novel relationships between genome 3D structure and oncogenic processes.

FKBP10-inhibited β-catenin ubiquitination in the malignant transformation of breast epithelial cells.

e12580 Background: Current model of breast cancer (BCa) progression suggests a linear and three-stage process from normal epithelial tissue to ductal carcinoma in situ (DCIS), and then invasive ductal carcinoma (IDC). Since the detailed mechanisms for BCa oncogenesis remain largely unclear and there is still a lack of root-cause level interventions, exploring the underlying molecular alterations during three-stage process of BCa carcinogenesis is urgently needed. Methods: Three-stage specimens from the very same patients were collected. High-throughput sequencing was applied to obtain the differentially expressed genes among normal, DCIS, and IDC samples. Expression level and prognostic value of the selected gene were evaluated by PCR, western blot, tissue chip, and bioinformatics analysis. MCF10DCIS and MCF10Ca cell lines were respectively used to establish DCIS and IDC tumor models in breast epithelial cell-specific gene knockout mice. Patient-matched DCIS and IDC organoids were also constructed. Signaling pathways associated with malignant transformation of breast epithelial cells were detected by using high-throughput sequencing in mouse models and organoids. Protein binding was evaluated by protein mass spectrometry and co-immunoprecipitation. Results: By using high-throughput sequencing, we found that FKBP10 levels were gradually increased in three-stage specimens from the very same patients and were associated with poor prognosis. By establishing DCIS and IDC models in FKBP10flox/floxCre+/- and FKBP10flox/floxCre-/- control mice, we confirmed that FKBP10 deficiency could suppress BCa formation. Besides, FKBP10 knockdown could reduce oncogenic potentials in patient-matched DCIS and IDC organoids. Wnt/β-catenin signaling pathway was related to malignant transformation of breast epithelial cells, when MCF10DCIS+FKBP10flox/floxCre+/- model, MCF10Ca+FKBP10flox/floxCre+/- model, FKBP10-knockdown DCIS and IDC organoids, and their corresponding controls were comprehensively compared. Moreover, FKBP10 was found to inhibit β-catenin ubiquitination with the help of deubiquitinase Trim13, thus activating Wnt/β-catenin signaling pathway and accelerating malignant transformation of breast epithelial cells. Increased level of β-catenin was able to promote FKBP10 expression in a feedback loop. We also confirmed that exosome-loaded FKBP10-targeting peptide could suppress tumor growth both in vitro and in vivo. Conclusions: FKBP10-inhibited β-catenin ubiquitination was responsible in the malignant transformation from normal tissue to DCIS, and then IDC. Our study would uncover novel regulatory mechanisms of FKBP10 in three-stage process of BCa carcinogenesis and provide a new therapeutic approach for clinical treatment.

Rare subclonal sequencing of breast cancers indicates putative metastatic driver mutations are predominately acquired after dissemination

BackgroundEvolutionary models of breast cancer progression differ on the extent to which metastatic potential is pre-encoded within primary tumors. Although metastatic recurrences often harbor putative driver mutations that are not detected in their antecedent primary tumor using standard sequencing technologies, whether these mutations were acquired before or after dissemination remains unclear.MethodsTo ascertain whether putative metastatic driver mutations initially deemed specific to the metastasis by whole exome sequencing were, in actuality, present within rare ancestral subclones of the primary tumors from which they arose, we employed error-controlled ultra-deep sequencing (UDS-UMI) coupled with FFPE artifact mitigation by uracil-DNA glycosylase (UDG) to assess the presence of 132 “metastasis-specific” mutations within antecedent primary tumors from 21 patients. Maximum mutation detection sensitivity was ~1% of primary tumor cells. A conceptual framework was developed to estimate relative likelihoods of alternative models of mutation acquisition.ResultsThe ancestral primary tumor subclone responsible for seeding the metastasis was identified in 29% of patients, implicating several putative drivers in metastatic seeding including LRP5 A65V and PEAK1 K140Q. Despite this, 93% of metastasis-specific mutations in putative metastatic driver genes remained undetected within primary tumors, as did 96% of metastasis-specific mutations in known breast cancer drivers, including ERRB2 V777L, ESR1 D538G, and AKT1 D323H. Strikingly, even in those cases in which the rare ancestral subclone was identified, 87% of metastasis-specific putative driver mutations remained undetected. Modeling indicated that the sequential acquisition of multiple metastasis-specific driver or passenger mutations within the same rare subclonal lineage of the primary tumor was highly improbable.ConclusionsOur results strongly suggest that metastatic driver mutations are sequentially acquired and selected within the same clonal lineage both before, but more commonly after, dissemination from the primary tumor, and that these mutations are biologically consequential. Despite inherent limitations in sampling archival primary tumors, our findings indicate that tumor cells in most patients continue to undergo clinically relevant genomic evolution after their dissemination from the primary tumor. This provides further evidence that metastatic recurrence is a multi-step, mutation-driven process that extends beyond primary tumor dissemination and underscores the importance of longitudinal tumor assessment to help guide clinical decisions.

Design of a mucin-selective protease for targeted degradation of cancer-associated mucins.

Targeted protein degradation is an emerging strategy for the elimination of classically undruggable proteins. Here, to expand the landscape of targetable substrates, we designed degraders that achieve substrate selectivity via recognition of a discrete peptide and glycan motif and achieve cell-type selectivity via antigen-driven cell-surface binding. We applied this approach to mucins, O-glycosylated proteins that drive cancer progression through biophysical and immunological mechanisms. Engineering of a bacterial mucin-selective protease yielded a variant for fusion to a cancer antigen-binding nanobody. The resulting conjugate selectively degraded mucins on cancer cells, promoted cell death in culture models of mucin-driven growth and survival, and reduced tumor growth in mouse models of breast cancer progression. This work establishes a blueprint for the development of biologics that degrade specific protein glycoforms on target cells.

PTHrP Regulates Fatty Acid Metabolism via Novel lncRNA in Breast Cancer Initiation and Progression Models.

Parathyroid hormone-related peptide (PTHrP) is the primary cause of malignancy-associated hypercalcemia (MAH). We previously showed that PTHrP ablation, in the MMTV-PyMT murine model of breast cancer (BC) progression, can dramatically prolong tumor latency, slow tumor growth, and prevent metastatic spread. However, the signaling mechanisms using lineage tracing have not yet been carefully analyzed. Here, we generated Pthrpflox/flox; Cre+ mT/mG mice (KO) and Pthrpwt/wt; Cre+ mT/mG tumor mice (WT) to examine the signaling pathways under the control of PTHrP from the early to late stages of tumorigenesis. GFP+ mammary epithelial cells were further enriched for subsequent RNA sequencing (RNAseq) analyses. We observed significant upregulation of cell cycle signaling and fatty acid metabolism in PTHrP WT tumors, which are linked to tumor initiation and progression. Next, we observed that the expression levels of a novel lncRNA, GM50337, along with stearoyl-Coenzyme A desaturase 1 (Scd1) are significantly upregulated in PTHrP WT but not in KO tumors. We further validated a potential human orthologue lncRNA, OLMALINC, together with SCD1 that can be regulated via PTHrP in human BC cell lines. In conclusion, these novel findings could be used to develop targeted strategies for the treatment of BC and its metastatic complications.

ADAMTS3 restricts cancer invasion in models of early breast cancer progression through enhanced fibronectin degradation

Proteases have long been associated with cancer progression, due to their ability to facilitate invasion upon matrix remodelling. However, proteases are not simply degraders of the matrix, but also play fundamental roles in modulating cellular behaviour through the proteolytic processing of specific substrates. Indeed, proteases can elicit both pro- and anti- tumorigenic effects depending on context. Using a heterocellular spheroid model of breast cancer progression, we demonstrate the repressive function of myoepithelial ADAMTS3, with its loss directing myoepithelial-led invasion of luminal cells through a physiologically relevant matrix. Degradomic analysis, using terminal amine isotopic labelling of substrates (TAILS), combined with functional assays, implicate ADAMTS3 as a mediator of fibronectin degradation. We show further that loss of ADAMTS3 enhances levels of fibronectin in the microenvironment, promoting invasion through canonical integrin α5β1 activation. Our data highlight a tumour suppressive role for ADAMTS3 in early stage breast cancer, and contribute to the growing evidence that proteases can restrain cancer progression.

Spatiotemporal higher-order chromatin landscape of human histone gene clusters at histone locus bodies during the cell cycle in breast cancer progression

Human Histone Locus Bodies (HLBs) are nuclear subdomains comprised of clustered histone genes that are coordinately regulated throughout the cell cycle. We addressed temporal-spatial higher-order genome organization for time-dependent chromatin remodeling at HLBs that supports control of cell proliferation. Proximity distances of specific genomic contacts within histone gene clusters exhibit subtle changes during the G1 phase in MCF10 breast cancer progression model cell lines. This approach directly demonstrates that the two principal histone gene regulatory proteins, HINFP (H4 gene regulator) and NPAT, localize at chromatin loop anchor-points, denoted by CTCF binding, supporting the stringent requirement for histone biosynthesis to package newly replicated DNA as chromatin. We identified a novel enhancer region located ∼ 2 MB distal to histone gene sub-clusters on chromosome 6 that consistently makes genomic contacts with HLB chromatin and is bound by NPAT. During G1 progression the first DNA loops form between one of three histone gene sub-clusters bound by HINFP and the distal enhancer region. Our findings are consistent with a model that the HINFP/NPAT complex controls the formation and dynamic remodeling of higher-order genomic organization of histone gene clusters at HLBs in early to late G1 phase to support transcription of histone mRNAs in S phase.

Abstract P5-13-01: TUMOR PROGRESSION MODEL IN BREAST CANCER WITH BRAIN METASTASIS

Abstract INTRODUCTION: Breast cancer are the main cause of related deaths cancer among women, corresponding to 25% of new cases each year. When diagnosed at early stages, it has an overall five-year survival rate of up to 90%. However, in more advanced stages, survival is reduced to about 24%, with 90% of women in stage IV dying as a result of complications related to metastases. Considering that brain metastasis is an unfavorable prognostic site, and the identification of genetic-molecular profiles in primary tumors and in metastatic sites are a subject poorly described in the literature, we understand that the identification of mutational profiles may contribute to elucidate the genetic-molecular mechanisms associated with tumor progression. AIM: The aim of this study was to identify clonal and subclonal driver mutations that lead to evolution of metastatic clones from a breast cancer progression model. MATERIAL AND METHODS: For tumor progression model, automated extraction of DNA from buffy coat and paraffin samples of breast tumors and paired brain metastases (n=9) was performed. In the present work, we used a subclonal reconstruction model based on the combination of machine learning and population genetics concepts. This proposal is based on the frequency spectrum of each somatic mutation (SNVs or indels), considering VAF (Variant Allele Frequency - ratio of mapped reads of the mutant allele) in relation to the coverage of variant locus, as known as CCF (Cancer Cell Fraction). CCF is defined as the proportion of neoplastic cells that have a certain set of mutations and then is normalized considering the sample purity and the segments with changes in number of DNA copies (Copy Number Alterations). Then, a statistical model based on finite Dirichlet mixtures with mixed distributions is applied. In this model, Beta components capture clonal expansions and population genetics concepts were applied to mutant alleles in each population considering principles of cancer evolution. Finally, confidence was computed using both parametric and non-parametric bootstraps. The functions for building the model are implemented at https://caravagnalab.github.io/mobster/, and for visualization and construction of graphs, packages in R statistical-mathematical environment were used, such as ggplot2, sads, cli, clisymbols, cowplot, crayon, ctree, dndscv, dplyr, magrittr, reshape2, and tidyr. RESULTS: With this model was possible to identify the distribution of clones according to somatic alterations in patients with breast cancer and brain metastasis. It was possible to observe common patterns, such as alterations of the SF3B1 gene as a common ancestral clone in both conditions and the frequency of the AKT1 gene in a subclonal condition. Other genes relevant to breast cancer carcinogenesis, such as PIK3CA and TP53, are found in a different clonal hierarchical pattern between the two conditions. CONCLUSION: This data suggests a model of clonal evolution capable to identify which drivers clones and subclones are involved in the metastatic process. Citation Format: Muriele B. Varuzza, Adriane F. Evangelista, Cristiano P. Souza, Márcia C. Marques. TUMOR PROGRESSION MODEL IN BREAST CANCER WITH BRAIN METASTASIS [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-13-01.

Abstract B012: Exosome microRNAs are progressively altered and contribute to invasive progression of ductal carcinoma in situ

Abstract Ductal carcinoma in situ (DCIS) is a benign breast condition that increases the risk of invasive breast cancer (IBC). Clinically, not all DCIS will progress to IBC and the major drivers of progression are not well understood. The role of small extracellular vesicles, also known as exosomes, is well described in advanced cancers, however, little is known about their role in the invasive progression of in situ breast disease. In this study, we sought to evaluate the role of exosomes and their RNA contents in the progression of DCIS, using the MCF10 isogenic series, in vitro assays and the mouse intraductal model of breast cancer progression. We observed a significant increase of exosome loading of microRNAs (miRNAs) between normal and benign states that increased with each stage. We demonstrate that pro-invasive programs were attenuated when overall exosome and miRNA secretion was inhibited by Rab27A and Dicer knockdown, respectively. Comparisons of exosome miRNA expression changes revealed the greatest differential expression between in situ and invasive cells, with miR-205 being the most significantly different, suggesting a mechanistic role for exosome miRNAs in invasive progression. Modulation of miR-205 levels affected epithelial to mesenchymal transition and the invasive programs in vitro. Progression of DCIS in vivo resulted in increased levels of specific circulating exosome miRNAs, while inhibition of exosome secretion (Rab27A knockdown) attenuated invasive progression and reduced circulating levels of exosome miRNAs. These observations suggest that exosome miRNAs are differentially loaded and expressed, and promote the invasive progression of DCIS and that circulating exosome miRNAs are a potential source of biomarkers of the DCIS to invasive transition. Citation Format: Bethany Hannafon, Samrita Dogra, Sugantha Priya Elayapillai, James Lausen, Matthew Bruns, Fariba Behbod, Amy Gin, Chao Xu, Roy Zhang, Wei-Qun Ding. Exosome microRNAs are progressively altered and contribute to invasive progression of ductal carcinoma in situ [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr B012.

A natural history and copula-based joint model for regional and distant breast cancer metastasis.

The few existing statistical models of breast cancer recurrence and progression to distant metastasis are predominantly based on multi-state modelling. While useful for summarising the risk of recurrence, these provide limited insight into the underlying biological mechanisms and have limited use for understanding the implications of population-level interventions. We develop an alternative, novel, and parsimonious approach for modelling latent tumour growth and spread to local and distant metastasis, based on a natural history model with biologically inspired components. We include marginal sub-models for local and distant breast cancer metastasis, jointly modelled using a copula function. Different formulations (and correlation shapes) are allowed, thus we can incorporate and directly model the correlation between local and distant metastasis flexibly and efficiently. Submodels for the latent cancer growth, the detection process, and screening sensitivity, together with random effects to account for between-patients heterogeneity, are included. Although relying on several parametric assumptions, the joint copula model can be useful for understanding - potentially latent - disease dynamics, obtaining patient-specific, model-based predictions, and studying interventions at a population level, for example, using microsimulation. We illustrate this approach using data from a Swedish population-based case-control study of postmenopausal breast cancer, including examples of useful model-based predictions.

Surface cholesterol-enriched domains specifically promote invasion of breast cancer cell lines by controlling invadopodia and extracellular matrix degradation

Tumor cells exhibit altered cholesterol content. However, cholesterol structural subcellular distribution and implication in cancer cell invasion are poorly understood mainly due to difficulties to investigate cholesterol both quantitatively and qualitatively and to compare isogenic cell models. Here, using the MCF10A cell line series (non-tumorigenic MCF10A, pre-malignant MCF10AT and malignant MCF10CAIa cells) as a model of breast cancer progression and the highly invasive MDA-MB-231 cell line which exhibits the common TP53 mutation, we investigated if cholesterol contributes to cancer cell invasion, whether the effects are specific to cancer cells and the underlying mechanism. We found that partial membrane cholesterol depletion specifically and reversibly decreased invasion of the malignant cell lines. Those cells exhibited dorsal surface cholesterol-enriched submicrometric domains and narrow ER-plasma membrane and ER-intracellular organelles contact sites. Dorsal cholesterol-enriched domains can be endocytosed and reach the cell ventral face where they were involved in invadopodia formation and extracellular matrix degradation. In contrast, non-malignant cells showed low cell invasion, low surface cholesterol exposure and cholesterol-dependent focal adhesions. The differential cholesterol distribution and role in breast cancer cell invasion provide new clues for the understanding of the molecular events underlying cellular mechanisms in breast cancer.

Gene signature associated with resistance to fluvastatin chemoprevention for breast cancer

BackgroundAlthough targeting of the cholesterol pathway by statins prevents breast cancer development in mouse models, efficacy is not absolute. Therefore, the goal of this study is to investigate if the upregulation in the cholesterol biosynthesis pathway genes associates with response to statin chemoprevention and may potentially be used as response biomarkers.MethodsExpression of cholesterol biosynthesis pathway genes was initially derived from the RNA sequencing of MCF10A cell line- based breast cancer progression model system and subsequently validated by quantitative PCR assay. Response to fluvastatin was assessed in vitro using the MCF10A cell line model system, including a statin resistant cell line that was generated (MCF10.AT1-R), and measured using colony forming assays. In vivo efficacy of statin for chemoprevention was assessed in the SV40C3 TAg mouse model. Mammary tumors were identified by histologic analysis of the mammary glands. Mammary glands without histologic evidence of high-grade lesions (in situ and/or invasive carcinoma) were considered responsive to statin treatment.ResultsWe found more than 70% of a published multi-gene fluvastatin resistance signature to be significantly upregulated during breast cancer progression and inversely correlated with statin inhibition of cellular growth and proliferation. This inherent statin resistance gene signature was also largely shared with the signature of acquired resistance to fluvastatin in MCF10.AT1-R cell line model of acquired statin resistance. These inherent resistance genes and genes exclusive to acquired statin resistance map to steroid-, and terpenoid backbone- biosynthesis pathway. We found upregulation of ~ 80% of cholesterol biosynthesis pathway genes in the tumor bearing mammary glands of SV40 C3TAg transgenic mouse model of TNBC, suggesting the involvement of cholesterol biosynthesis pathway in resistance to statin chemoprevention in vivo. A panel of 13-genes from the pathway significantly associated with response to statin treatment, as did the expression level of HMGCR alone in a mouse model of breast cancer suggesting their utility to predict the efficacy of statin chemoprevention.ConclusionsHigh basal level, or restorative upregulation, in the cholesterol biosynthesis pathway genes appear to be strongly associated with resistance to statin chemoprevention for breast cancer and may serve as a biomarker to tailor statin treatment to individuals who are most likely to benefit.

Abstract P2-11-10: Heterogeneity of preneoplastic breast tissues drives efficacy of therapeutic agents

Abstract Introduction: Little is known about the evolution of proteomic aberrations during TNBC tumorigenesis and whether the timing of intervention for prevention and treatment during this tumorigenic progression impacts efficacy. We hypothesized that the targeted therapies for TNBC is likely to be context specific, with specific transition points where such therapy is likely to be most effective based on presence of proteomic aberrations. Methods: MCF10A cell line-based model of triple negative breast cancer progression was used. This model system comprises of non-cancer MCF10A (P), preneoplastic MCF10.AT1 and MCF10.NeoT, premalignant ductal carcinoma in situ MCF10.DCIS and invasive MCF10.CA1D cells. Global proteomic patterns were studied by performing RPPA assay. Growth inhibitory effects drugs were studied by measuring inhibition in cell proliferation by MTT assay. Results: RPPA analysis revealed a majority of protein alterations (n=48) were acquired early on during TNBC progression, specifically during normal to preneoplastic (atypia) transition, with only 6 aberrations acquired in later stages of tumorigenesis. To assess the effects of this heterogeneity on efficacy of treatment, we selected 2 aberrant pathways that were up-regulated early in the breast tumorigenesis and compared efficacy of targeted intervention to drugs without pathway selectivity. Targeting upstream oncogenic pathways with small molecule inhibitors (AKT pathway with LY294002- a PI3 kinase inhibitor and MEK pathway with MEK1/2 inhibitors PD032590 or GSK1120212) showed PI3K inhibitor to be equally effective in suppressing the cell proliferation in both preneoplastic and DCIS state, consistent with upregulation of the underlying AKT pathway aberration early in tumorigenesis. MEK pathway inhibitors were preferentially more effective in preneoplastic state than DCIS state, consistent with continued activation of MEK pathway in the DCIS state relative to preneoplastic state. Similarly, fluvastatin (a cholesterol lowering drug) was more effective in inhibiting the cell proliferation in preneoplastic cells relative to DCIS cells. However, a broad spectrum drug aspirin (an AMPK activator) that is known to have pleotropic effects showed no differential effects across the cell lines tested. Conclusions: The preponderance of proteomic alterations occur in the very earliest stages of TNBC tumorigenesis. Effectiveness of targeted drug therapies in this model of breast tumorigenesis correlated. with deregulation of the associated pathway. This implies that accounting for heterogeneity of the preneoplastic breast and stage of tumorigenic progression will be important when considering targeted strategies for prevention. Citation Format: Anjana Bhardwaj, Raniv Rojo, Zhenlin Ju, Jing Wang, Isabelle Bedrosian. Heterogeneity of preneoplastic breast tissues drives efficacy of therapeutic agents [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 P2-11-10.

The Value of EphB2 Receptor and Cognate Ephrin Ligands in Prognostic and Predictive Assessments of Human Breast Cancer.

Cell–cell communication proteins Eph and ephrin constitute the largest family of receptor tyrosine kinases (RTKs). They are distinguished by the fact that both receptors and ligands are membrane-bound, and both can drive intracellular signaling in their respective cells. Ever since these RTKs have been found to be involved in cancer development, strategies to target them therapeutically have been actively pursued. However, before this goal can be rationally achieved, the contributions of either Eph receptors or their ephrin ligands to cancer development and progression should be scrutinized in depth. To assess the clinical pertinence of this concern, we performed a systematic review and meta-analysis of the prognostic/predictive value of EphB2 and its multiple cognate ephrin ligands in breast cancer. We found that EphB2 has prognostic value, as indicated by the association of higher EphB2 expression levels with lower distant metastasis-free survival (DMFS), and the association of lower EphB2 expression levels with poorer relapse-free survival (RFS). We also found that higher EphB2 expression could be a prognostic factor for distant metastasis, specifically in the luminal subtypes of breast cancer. EFNB2 showed a marked correlation between higher expression levels and shorter DMFS. EFNA5 or EFNB1 overexpression is correlated with longer RFS. Increased EFNB1 expression is correlated with longer OS in lymph node (LN)-negative patients and the luminal B subtype. Higher levels of EFNB2 or EFNA5 are significantly correlated with shorter RFS, regardless of LN status. However, while this correlation with shorter RFS is true for EFNB2 in all subtypes except basal, it is also true for EFNA5 in all subtypes except HER2+. The analysis also points to possible predictive value for EphB2. In systemically treated patients who have undergone either endocrine therapy or chemotherapy, we found that higher expression of EphB2 is correlated with better rates of RFS. Bearing in mind the limitations inherent to any mRNA-based profiling method, we complemented our analysis with an immunohistochemical assessment of expression levels of both the EphB2 receptor and cognate ephrin ligands. We found that the latter are significantly more expressed in cancers than in normal tissues, and even more so in invasive and metastatic samples than in ductal carcinoma in situ (DCIS). Finally, in an in vitro cellular model of breast cancer progression, based on H-Ras-transformation of the MCF10A benign mammary cell line, we observed dramatic increases in the mRNA expression of EphB2 receptor and EFNB1 and EFNB2 ligands in transformed and invasive cells in comparison with their benign counterparts. Taken together, these data show the clinical validity of a model whereby EphB2, along with its cognate ephrin ligands, have dual anti- and pro-tumor progression effects. In so doing, they reinforce the necessity of further biological investigations into Ephs and ephrins, prior to using them in targeted therapies.

Bidirectional Regulatory Cross-Talk between Cell Context and Genomic Aberrations Shapes Breast Tumorigenesis.

Breast cancers are classified into five intrinsic subtypes and 10 integrative clusters based on gene expression patterns and genomic aberrations, respectively. Although the cell-of-origin, adaptive plasticity, and genomic aberrations shape dynamic transcriptomic landscape during cancer progression, how interplay between these three core elements governs obligatory steps for a productive cancer progression is unknown. Here, we used genetic ancestry-mapped immortalized breast epithelial cell lines generated from breast biopsies of healthy women that share gene expression profiles of luminal A, normal-like, and basal-like intrinsic subtypes of breast cancers and breast cancer relevant oncogenes to develop breast cancer progression model. Using flow cytometry, mammosphere growth, signaling pathway, DNA damage response, and in vivo tumorigenicity assays, we provide evidence that establishes cell context-dependent effects of oncogenes in conferring plasticity, self-renewal/differentiation, intratumor heterogeneity, and metastatic properties. In contrast, oncogenic aberrations, independent of cell context, shaped response to DNA damage-inducing agents. Collectively, this study reveals how the same set of genomic aberration can have distinct effects on tumor characteristics based on cell-of-origin of tumor and highlights the need to utilize multiple "normal" epithelial cell types to decipher oncogenic properties of a gene of interest. In addition, by creating multiple isogenic cell lines ranging from primary cells to metastatic variants, we provide resources to elucidate cell-intrinsic properties and cell-oncogene interactions at various stages of cancer progression. IMPLICATIONS: Our findings demonstrate that how an interplay between the normal cell type that encountered genomic aberrations and type of genomic aberration influences heterogeneity, self-renewal/differentiation, and tumor properties including propensity for metastasis.

Abstract 2589: Gene signature of fluvastatin resistance for prevention of breast cancer and predicting resistance to statins

Abstract Introduction: Cholesterol biosynthesis pathway is highly regulated and inhibition of the pathway with statins is known to cause restorative upregulation of several genes in the pathway. The goal of this study is to investigate if the statin mediated upregulation in the cholesterol biosynthesis pathway genes associates with the resistance to fluvastatin in a model of hormonally insensitive breast cancer Methods: A published gene signature of statin resistance was validated 1) a cell line based model of breast cancer progression consisting of inherently fluvastatin sensitive and inherently fluvastatin resistant cell lines and also compared to our experimentally derived acquired signature of fluvastatin resistance using 2) an isogenic set of cell lines consisting of a fluvastatin sensitive cell line (MCF10.AT1), and an acquired resistant cell line (MCF10.AT- R) and lastly validated using 3) SV40 C3 tag, a mouse model of hormone receptor negative breast cancer. Clariom RNA profiling were processed and mined by IPA analysis to identify the fluvastatin resistance signature that were validated by qPCR. Fluvastatin resistance was determined in vitro by colony formation assay and in vivo in a mouse model of breast cancer. Results: We found more than 75% of the published 17 gene panel fluvastatin resistance gene signature (consisting of cholesterol biosynthesis pathway genes) to be significantly upregulated in an inherently resistant cell line, DCIS cell line, relative to fluvastatin sensitive preneoplastic, MCF10.AT1 cell line. We found this inherent statin resistance gene signature to be also relevant in the MCF10.AT-R resistant cells as we found 13 of these genes to map to top 3 upregulated pathways that are steroid biosynthesis, steroid hormone biosynthesis and terpenoid backbone biosynthesis pathway. Next, we tested if 17 gene statin resistance signature associates with presence of tumors in the mammary glands of fluvastatin treated mice and found upregulation of more than 50% of the genes in the resistance signature in the tumor bearing mammary glands. Lastly, we studied if a 10-day period of fluvastatin treatment to SV40C3 Tag mice, a spontaneous mouse model of breast cancer, can also trigger the upregulation of these cholesterol biosynthesis pathway genes and provide an early signal of statin resistance. These experiments showed that a 10-day period is not long enough to cause a feedback upregulation in steroid biosynthesis pathway genes and thus can't be used a surrogate timepoint to detect resistance to fluvastatin. Conclusions: Upregulation of multiple steroid biosynthesis pathway genes after fluvastatin treatment suggests an opportunity of dual targeting of the cholesterol biosynthesis pathway in order to sensitize the fluvastatin resistant breast cancer cells. Citation Format: Anjana Bhardwaj, Zhenlin Ju, Matthew Embury, Jing Wang, Isabelle Bedrosian. Gene signature of fluvastatin resistance for prevention of breast cancer and predicting resistance to statins [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2589.

CD44 alternative splicing senses intragenic DNA methylation in tumors via direct and indirect mechanisms.

DNA methylation (meDNA) is a modulator of alternative splicing, and splicing perturbations are involved in tumorigenesis nearly as frequently as DNA mutations. However, the impact of meDNA on tumorigenesis via splicing-mediated mechanisms has not been thoroughly explored. Here, we found that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1/3b undergo a partial epithelial to mesenchymal transition associated with increased CD44 variant exon skipping. These skipping events are directly mediated by the loss of intragenic meDNA and the chromatin factors MBD1/2/3 and HP1γ and are also linked to phosphorylation changes in elongating RNA polymerase II. The role of meDNA in alternative splicing was confirmed by using the dCas9/DNMT3b tool. We further tested whether the meDNA level could have predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes, mostly involved in splicing and signal transduction, are correlated with the local modulation of meDNA. Our observations suggest that, although DNA methylation has multiple avenues to affect alternative splicing, its indirect effect may also be mediated through alternative splicing isoforms of these meDNA sensors.

A Simulation Model of Breast Cancer Incidence, Progression, Diagnosis and Survival in India

For resource-constrained health systems, it becomes important to evaluate the cost-effectiveness of a breast cancer (BC) screening program prior to its real-world implementation. In this paper, we provide an overview of a new simulation model of BC incidence, progression, and diagnosis developed to maximize the cost-effectiveness of a BC screening program. We describe the development of the modules for BC incidence, tumor growth and diagnosis, and survival estimation of diagnosed patients. Incidence of BC is based on published risk factors and publicly available national cancer registry reports. Survival curves for diagnosed patients based on specific characteristics such as age group and stage of diagnosis are generated using a single published survival curve for the entire population and hazard ratios for each characteristic. Our simulation development approach can provide a model development template for researchers working to develop BC simulation models in other settings with data availability similar to our case.

Pan-cancer analysis reveals TAp63-regulated oncogenic lncRNAs that promote cancer progression through AKT activation

The most frequent genetic alterations across multiple human cancers are mutations in TP53 and the activation of the PI3K/AKT pathway, two events crucial for cancer progression. Mutations in TP53 lead to the inhibition of the tumour and metastasis suppressor TAp63, a p53 family member. By performing a mouse-human cross species analysis between the TAp63 metastatic mammary adenocarcinoma mouse model and models of human breast cancer progression, we identified two TAp63-regulated oncogenic lncRNAs, TROLL-2 and TROLL-3. Further, using a pan-cancer analysis of human cancers and multiple mouse models of tumour progression, we revealed that these two lncRNAs induce the activation of AKT to promote cancer progression by regulating the nuclear to cytoplasmic translocation of their effector, WDR26, via the shuttling protein NOLC1. Our data provide preclinical rationale for the implementation of these lncRNAs and WDR26 as therapeutic targets for the treatment of human tumours dependent upon mutant TP53 and/or the PI3K/AKT pathway.