Biology subjects

Atkinson, J. M.

Publications and source records attributed to Atkinson, J. M..

14 recordsLinked to original sources

bioRxiv2026

Breast cancer ovarian metastases show increased activity of GPCR pathways

Treatment resistance and metastases occur in 10-20% of patients with invasive lobular carcinoma (ILC), the most common special histological subtype of breast cancer. ILC metastasizes to the ovary more frequently than no special type (NST) tumors, also known as invasive ductal carcinoma (IDC). To characterize the genomic landscape of breast cancer ovarian metastases, we analyzed 15,613 local breast cancers, 22,010 non-ovarian metastases, and 246 ovarian metastases sequenced using FoundationOne(R)CDx or FoundationOne(R) assays. Ovarian metastases had enriched CDH1, PIK3CA, and TBX3 mutations and depleted TP53 and MYC alterations relative to local breast cancers, with additional depletion of ESR1 mutations compared to non-ovarian metastases. CDH1 mutations were less frequent in ovarian metastases (47%) than local ILC (81.3%), with reduced 16q loss (64% vs 84%), indicating that ovarian metastases also arise from non-ILC tumors. We extended these findings to a UPMC cohort of 27 ovarian metastases (13 ILC, 8 IDC, 6 mixed ductal-lobular carcinoma) with patient-matched primary tumors in most cases. In both cohorts, patients with ovarian metastases were significantly younger than those with other metastatic sites. In the UPMC cohort, the most frequent mutations were in PIK3CA, CDH1, KMT2C, FOXA1, and RUNX1. Transcriptomic analysis identified upregulated G protein-coupled receptor (GPCR) pathways, including metabotropic glutamate receptor signaling. Functional studies showed that calcium-sensing receptor (CaSR), a GPCR overexpressed in ovarian metastases, drives MEK/ERK-dependent migration and F-actin reorganization in ILC cell lines, enhanced by estrogen and blocked by calcilytic, MEK, or anti- estrogen treatment. Our findings inform future therapeutic targeting of ovarian metastasis.

cancer biology↗

bioRxiv2025

Identification and Credentialing of Patient Derived Xenograft Models of Invasive Lobular Breast Carcinoma using Multi-omics and Histopathology assessment

Breast cancer is a heterogeneous disease with numerous histological subtypes. Invasive lobular cancer (ILC) is the most common special subtype, accounting for 10-15% of all breast cancers. The pathognomonic feature of ILC is the loss of E-cadherin (CDH1), which leads to a unique single-file growth pattern of discohesive cells. Although ILCs show better prognostic factors than the most common No Special Type (NST) of breast cancer, patients with ILC have worse long-term outcomes, which is not well understood. In this study, we aimed to identify and characterize Patient-Derived Xenograft (PDX) models of ILC based upon the presence of truncating CDH1 mutations and/or low CDH1 mRNA expression among 128 human breast cancer PDX models. We selected 8 PDX models for validation using Immunohistochemical (IHC) analysis for E-Cadherin, p120, ER, PR, and HER2. We confirmed that seven of these PDX models are indeed ILC while one was identified as mixed NST-ILC PDX. Molecular analysis of the confirmed ILC PDX models showed enrichment of truncating CDH1 mutations, significantly lower levels of CDH1 mRNA expression and predominantly luminal subtypes compared to NST PDX models, in line with the molecular characteristics of human ILC disease. The commonly altered genes in the ILC PDX models included PIK3CA (57%), CDH1 (57%) and TP53 (57%) among others. Our study confirms and characterizes new ILC PDX models, offering valuable tools to advance our understanding of human ILC biology and support the development of innovative treatment strategies.

cancer biology↗

bioRxiv2025

Targeting RET in Brain Metastases from Estrogen Receptor Positive Breast Cancer

Brain metastases (BrM) occur in 10-15% of patients with estrogen receptor (ER)-positive breast cancer, and remain a significant clinical challenge. While current therapeutic paradigms, including surgical resection and systemic therapy, have efficacy in the management of BrM from ER+BC, median overall survival following the diagnosis of BrM is approximately 18 months. The limited efficacy of current therapies, along with a relative paucity of therapeutic options, highlight the urgent clinical need to identify druggable targets for BrM from ER+BC. We previously identified recurrent overexpression of the receptor tyrosine kinase RET in breast cancer BrM relative to patient-matched primary tumors. The principal ligand for RET, Glial Cell-Derived Neurotrophic Factor (GDNF), is predominately expressed in the brain, where it functions in the maintenance of neurons and multiple glial cell types. Here, we show that increased RET expression and activation correlates with brain metastasis in breast cancer patients. Further, we confirm the role of RET signaling in the promotion of metastatic properties in vitro, including tumor spheroid formation, migration, invasion, and transformation. Using RET-overexpressing and RET-deficient models in four ER+BC cell lines, we demonstrate that RET overexpression enhances activation of RET downstream targets, such as ERK and AKT, and promotes metastatic phenotypes in in vitro, ex vivo and in vivo assays. Importantly, we demonstrate that the phenotypic effects of GDNF-mediated RET signaling can be abrogated by administration of Pralsetinib, a highly potent RET-selective kinase inhibitor. Using an ex vivo mouse brain slice co-culture model, we further demonstrate that RET overexpression in breast cancer cell lines potentiates their colonization and invasion into the brain tissues, which are reduced in RET-knockdown cell lines. RET overexpression facilitates brain colonization of breast cancer cells in vivo and is associated with reduced survival. Using proteomic analyses, we identify novel GDNF-mediated signaling pathways in breast cancer cell line models, including PLC{gamma}, P70S6K, STAT3 and CREB. RET-mediated activation of this range of downstream targets is sensitive to inhibition by Pralsetinib. In sum, this study identifies and characterizes RET as a targetable driver of breast cancer brain metastasis, and further describes the action of Pralsetinib in ER-positive breast cancer cell lines, providing insights to improve current targeted therapies for patients with breast cancer brain metastasis. Bullet pointsO_LIRET expression and activation are increased in breast cancer brain metastases. C_LIO_LIPralsetinib potently inhibits RET activation in RET overexpressing and in non-overexpressing breast cancer cell line models. C_LIO_LIRET overexpression and activation confers pro-metastatic properties including cell proliferation, colonization, migration and invasion in vitro and in ex vivo brain co-culture and enhances brain colonization of breast cancer cells in vivo. C_LIO_LIGDNF increases activation of ERK1/2, AKT, PCL{gamma}, P70S6K, STAT3, and CREB in RET overexpressing MCF-7 cells. C_LIO_LICREB or P70S6K knockdown attenuates GDNF stimulated activation of RET downstream targets and cell migration. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/647085v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@1628735org.highwire.dtl.DTLVardef@19ad565org.highwire.dtl.DTLVardef@2fdcc2org.highwire.dtl.DTLVardef@468233_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Systemic and local chronic inflammation and hormone disposition promote a tumor-permissive environment for breast cancer in older women

Estrogen receptor positive (ER+) breast cancer is the most common subtype of breast cancer and is an age-related disease. The peak incidence of diagnosis occurs around age 70, even though these post-menopausal patients have low circulating levels of estradiol (E2). Despite the hormone sensitivity of age-related tumors, we have a limited understanding of the interplay between systemic and local hormones, chronic inflammation, and immune changes that contribute to the growth and development of these tumors. Here, we show that aged F344 rats treated with the dimethylbenz(a)anthracene / medroxyprogestrone acetate (DMBA/MPA) carcinogen develop more tumors at faster rates than their younger counterparts, suggesting that the aged environment promotes tumor initiation and impacts growth. Single-nuclei RNA-seq (snRNA-seq) of the tumors showed broad local immune dysfunction that was associated with circulating chronic inflammation. Across a broad cohort of specimens from patients with ER+ breast cancer and age-matched donors of normal breast tissue, we observe that even with an estrone (E1)-predominant estrogen disposition in the systemic circulation, tumors in older patients increase HSD17B7 expression to convert E1 to E2 in the tumor microenvironment (TME) and have local E2 levels similar to pre-menopausal patients. Concurrently, trackable increases in several chemokines, defined most notably by CCL2, promote a chronically inflamed but immune dysfunctional TME. This unique milieu in the aged TME, characterized by high local E2 and chemokine-enriched chronic inflammation, promotes both accumulation of tumor-associated macrophages (TAMs), which serve as signaling hubs, as well as polarization of TAMs towards a CD206+/PD-L1+, immunosuppressive phenotype. Pharmacologic targeting of estrogen signaling (either by HSD17B7 inhibition or with fulvestrant) and chemokine inflammation both decrease local E2 and prevent macrophage polarization. Overall, these findings suggest that chronic inflammation and hormonal disposition are critical contributors to the age-related nature of ER+ breast cancer development and growth and offer potential therapeutic insight to treat these patients. Translational SummaryWe uncover the unique underpinnings establishing how the systemic host environment contributes to the aged breast tumor microenvironment, characterized by high local estradiol and chronic inflammation with immune dysregulation, and show that targeting avenues of estrogen conversion and chronic inflammation work to restore anti-tumor immunity. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/616978v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1ecd90borg.highwire.dtl.DTLVardef@9d7ac8org.highwire.dtl.DTLVardef@e7fb74org.highwire.dtl.DTLVardef@2da524_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2023

The invasive lobular cancer cell line encyclopedia (ICLE) for studying potential biomarkers and exploring therapeutic opportunities

Invasive lobular carcinoma (ILC), the most common histological "special type", accounts for [~]10-15% of all BC diagnoses, is characterized by unique features such as E-cadherin loss/deficiency, lower grade, hormone receptor positivity, larger diffuse tumors, and specific metastatic patterns. Despite ILC being acknowledged as a disease with distinct biology that necessitates specialized and precision medicine treatments, the further exploration of its molecular alterations with the goal of discovering new treatments has been hindered due to the scarcity of well-characterized cell line models for studying this disease. To address this, we generated the ILC Cell Line Encyclopedia (ICLE), providing a comprehensive multi-omic characterization of ILC and ILC-like cell lines. Using consensus multi-omic subtyping, we confirmed luminal status of previously established ILC cell lines and uncovered additional ILC/ILC-like cell lines with luminal features for modeling ILC disease. Furthermore, most of these luminal ILC/ILC-like cell lines also showed RNA and copy number similarity to ILC patient tumors. Similarly, ILC/ILC-like cell lines also retained molecular alterations in key ILC genes at similar frequency to both primary and metastatic ILC tumors. Importantly, ILC/ILC-like cell lines recapitulated the CDH1 alteration landscape of ILC patient tumors including enrichment of truncating mutations in and biallelic inactivation of CDH1 gene. Using whole-genome optical mapping, we uncovered novel genomic-rearrangements including novel structural variations in CDH1 and functional gene fusions and characterized breast cancer specific patterns of chromothripsis in chromosomes 8, 11 and 17. In addition, we systematically analyzed aberrant DNAm events and integrative analysis with RNA expression revealed epigenetic activation of TFAP2B - an emerging biomarker of lobular disease that is preferentially expressed in lobular disease. Finally, towards the goal of identifying novel druggable vulnerabilities in ILC, we analyzed publicly available RNAi loss of function breast cancer cell line datasets and revealed numerous putative vulnerabilities cytoskeletal components, focal adhesion and PI3K/AKT pathway in ILC/ILC-like vs NST cell lines. In summary, we addressed the lack of suitable models to study E-cadherin deficient breast cancers by first collecting both established and putative ILC models, then characterizing them comprehensively to show their molecular similarity to patient tumors along with uncovering their novel multi-omic features as well as highlighting putative novel druggable vulnerabilities. Not only we expand the array of suitable E-cadherin deficient cell lines available for modelling human-ILC disease but also employ them for studying epigenetic activation of a putative lobular biomarker as well as identifying potential druggable vulnerabilities for this disease towards enabling precision medicine research for human-ILC.

cancer biology↗

bioRxiv2023

ESR1 fusion proteins invoke breast cancer subtype-dependent enrichment of ligand independent pro-oncogenic signatures and phenotypes

Breast cancer is a leading cause of female mortality and despite advancements in diagnostics and personalized therapeutics, metastatic disease largely remains incurable due to drug resistance. Fortunately, identification of mechanisms of therapeutic resistance have rapidly transformed our understanding of cancer evasion and is enabling targeted treatment regimens. When the druggable estrogen receptor (ER, ESR1), expressed in two-thirds of all breast cancer, is exposed to endocrine therapy, there is risk of somatic mutation development in approximately 30% of cases and subsequent treatment resistance. A more recently discovered mechanism of ER mediated endocrine resistance is the expression of ER fusion proteins. ER fusions, which retain the proteins DNA binding domain, harbor ESR1 exons 1-6 fused to an in-frame gene partner resulting in loss of the 3 ER ligand binding domain (LBD). In this report we demonstrate that in no-special type (NST) and invasive lobular carcinoma (ILC) cell line models, ER fusion proteins exhibit robust hyperactivation of canonical ER signaling pathways independent of the ligand estradiol or anti-endocrine therapies such as Fulvestrant and Tamoxifen. We employ cell line models stably overexpressing ER fusion proteins with concurrent endogenous ER knockdown to minimize the influence of endogenous wildtype ER. Cell lines exhibited shared transcriptomic enrichment in pathways known to be drivers of metastatic disease, notably the MYC pathway. The heterogeneous 3 fusion partners, particularly transcription factors SOX9 and YAP1, evoked varying degrees of transcriptomic and cistromic activity that translated into unique phenotypic readouts. Herein we report that cell line activity is subtype-, fusion-, and assay-specific suggesting that the loss of the LBD, the 3 fusion partner, and the cellular landscape all influence fusion activity. Therefore, it will be critical to generate additional data on frequency of the ER fusions, in the context of the clinicopathological features of the tumor. SignificanceER fusion proteins exhibit diverse mechanisms of endocrine resistance in breast cancer cell lines representing the no special type (NST) and invasive lobular cancer (ILC) subtypes. Our emphasize upon both the shared and unique cellular adaptations imparted by ER fusions offers the foundation for further translational research and clinical decision making.

cancer biology↗

bioRxiv2023

WCRC-25: A novel luminal Invasive Lobular Carcinoma cell line model

Breast cancer is categorized by the molecular and histologic presentation of the tumor, with the major histologic subtypes being No Special Type (NST) and Invasive Lobular Carcinoma (ILC). ILC are characterized by growth in a single file discohesive manner with stromal infiltration attributed to their hallmark pathognomonic loss of E-cadherin (CDH1). Few ILC cell line models are available to researchers. Here we report the successful establishment and characterization of a novel ILC cell line, WCRC-25, from a metastatic pleural effusion from a postmenopausal Caucasian woman with metastatic ILC. WCRC-25 is an ER-negative luminal epithelial ILC cell line with both luminal and Her2-like features. It exhibits anchorage independent growth and haptotactic migration towards Collagen I. Sequencing revealed a CDH1 Q706* truncating mutation, together with mutations in FOXA1, CTCF, BRCA2 and TP53, which were also seen in a series of metastatic lesions from the patient. Copy number analyses revealed amplification and deletion of genes frequently altered in ILC while optical genome mapping revealed novel structural rearrangements. RNA-seq analysis comparing the primary tumor, metastases and the cell line revealed signatures for cell cycle progression and receptor tyrosine kinase signaling. To assess targetability, we treated WCRC-25 with AZD5363 and Alpelisib confirming WCRC-25 as susceptible to PI3K/AKT signaling inhibition as predicted by our RNA sequencing analysis. In conclusion, we report WCRC-25 as a novel ILC cell line with promise as a valuable research tool to advance our understanding of ILC and its therapeutic vulnerabilities. Financial supportThe work was in part supported by a Susan G Komen Leadership Grant to SO (SAC160073) and NCI R01 CA252378 (SO/AVL). AVL and SO are Komen Scholars, Hillman Foundation Fellows and supported by BCRF. This project used the UPMC Hillman Cancer Center and Tissue and Research Pathology/Pitt Biospecimen Core shared resource which is supported in part by award P30CA047904. This research was also supported in part by the University of Pittsburgh Center for Research Computing, RRID:SCR_022735, through the resources provided. Specifically, this work used the HTC cluster, which is supported by NIH award number S10OD028483. Finally, partial support was provided by the Magee-Womens Research Institute and Foundation, The Shear Family Foundation, and The Metastatic Breast Cancer Network.

cancer biology↗

bioRxiv2023

Spatial molecular profiling of mixed invasive ductal-lobular breast cancers reveals heterogeneity in intrinsic molecular subtypes, oncogenic signatures and actionable driver mutations

Mixed invasive ductal and lobular carcinoma (MDLC) is a rare histologic subtype of breast cancer displaying both E-cadherin positive ductal and E-cadherin negative lobular morphologies within the same tumor, posing challenges with regard to anticipated clinical management. It remains unclear whether these distinct morphologies also have distinct biology and risk of recurrence. Our spatially-resolved transcriptomic, genomic, and single-cell profiling revealed clinically significant differences between ductal and lobular tumor regions including distinct intrinsic subtype heterogeneity (e.g., MDLC with TNBC/basal ductal and ER+/luminal lobular regions), distinct enrichment of senescence/dormancy and oncogenic (ER and MYC) signatures, genetic and epigenetic CDH1 inactivation in lobular, but not ductal regions, and single-cell ductal and lobular sub-populations with unique oncogenic signatures further highlighting intra-regional heterogeneity. Altogether, we demonstrated that the intra-tumoral morphological/histological heterogeneity within MDLC is underpinned by intrinsic subtype and oncogenic heterogeneity which may result in prognostic uncertainty and therapeutic dilemma. SignificanceMDLC displays both ductal and lobular tumor regions. Our multi-omic profiling approach revealed that these morphologically distinct tumor regions harbor distinct intrinsic subtypes and oncogenic features that may cause prognostic uncertainty and therapeutic dilemma. Thus histopathological/molecular profiling of individual tumor regions may guide clinical decision making and benefit patients with MDLC, particularly in the advanced setting where there is increased reliance on next generation sequencing.

cancer biology↗

bioRxiv2022

Loss of E-cadherin Induces IGF1R Activation Revealing a Targetable Pathway in Invasive Lobular Breast Carcinoma

No Special Type breast cancer (NST; commonly known as Invasive Ductal Carcinoma (IDC)) and Invasive Lobular Carcinoma (ILC) are the two major histological subtypes of breast cancer with significant differences in clinicopathological and molecular characteristics. The defining pathognomonic feature of ILC is loss of cellular adhesion protein, E-cadherin (CDH1). We have previously shown that E-cadherin functions as a negative regulator of the Insulin-like Growth Factor 1 Receptor (IGF1R) and propose that E-cadherin loss in ILC sensitizes cells to growth factor signaling which thus alters their sensitivity to growth factor signaling inhibitors and their downstream activators. To investigate this potential therapeutic vulnerability, we generated CRISPR-mediated CDH1 knockout (CDH1 KO) IDC cell lines (MCF7, T47D, ZR75.1) to uncover the mechanism by which loss of E-cadherin results in IGF pathway activation. CDH1 KO cells demonstrated enhanced invasion and migration that was further elevated in response to IGF1, serum and Collagen I. CDH1 KO cells exhibited increased sensitivity to IGF resulting in elevated downstream signaling. Despite minimal differences in membranous IGF1R levels between wildtype (WT) and CDH1 KO cells, significantly higher ligand-receptor interaction was observed in the CDH1 KO cells, potentially conferring enhanced downstream signaling activation. Critically, increased sensitivity to IGF1R, PI3K, Akt and MEK inhibitors was observed in CDH1 KO cells and ILC patient-derived organoids, suggesting that these targets require further exploration in ILC treatment and that CDH1 loss may be exploited as a biomarker of response for patient stratification.

cancer biology↗

bioRxiv2021

Hotspot ESR1 mutations are multimodal and contextual drivers of breast cancer metastasis

Constitutively active estrogen receptor- (ER/ESR1) mutations have been identified in approximately one third of ER+ metastatic breast cancer. Although these mutations are known mediators of endocrine resistance, their potential role in promoting metastatic disease has not yet been mechanistically addressed. In this study, we show the presence of ESR1 mutations exclusively in distant, but not local recurrences. In concordance with transcriptomic profiling of ESR1 mutant tumors, genome-edited Y537S and D538G cell models have a reprogrammed cell adhesive gene network via alterations in desmosome/gap junction genes and the TIMP3/MMP axis, which functionally confers enhanced cell-cell contacts while decreased cell-ECM adhesion. Context-dependent migratory phenotypes revealed co-targeting of Wnt and ER as vulnerability. Mutant ESR1 exhibits non-canonical regulation of several metastatic pathways including secondary transactivation and de novo FOXA1-driven chromatin remodeling. Collectively, our data supports evidence for ESR1 mutation-driven metastases and provides insight for future preclinical therapeutic strategies. SignificanceContext and allele-dependent transcriptome and cistrome reprogramming in genome-edited ESR1 mutation cell models elicit diverse metastatic phenotypes, including but not limited to alterations in cell adhesion and migration. The gain-of-function mutations can be pharmacologically targeted, and thus may be key components of novel therapeutic treatment strategies for ER-mutant metastatic breast cancer.

cancer biology↗

bioRxiv2020

ESR1 mutant breast cancers show elevated basal cytokeratins and immune activation

Estrogen receptor alpha (ER/ESR1) is mutated in 30-40% of endocrine resistant ER-positive (ER+) breast cancer. ESR1 mutations cause ligand-independent growth and increased metastasis in vivo and in vitro. Despite the distinct clinical features and changes in therapeutic response associated with ESR1 mutations, there are no data about their potential role in intrinsic subtype switching. Applying four luminal and basal gene set pairs, ESR1 mutant cell models and clinical samples showed a significant enrichment of basal subtype markers. Among them, the six basal cytokeratins (BCKs) were the most enriched genes. Induction of BCKs was independent of ER binding and instead associated with chromatin reprogramming centered around a progesterone receptor-orchestrated topological associated domain at the KRT14/16/17 genomic region. Unexpectedly, high BCK expression in ER+ primary breast cancer is associated with good prognosis, and these tumors show enriched activation of a number of immune pathways, a distinctive feature shared with ESR1 mutant tumors. S100A8 and S100A9 were among the most highly induced immune mediators shared between high-BCKs ER+ and ESR1 mutant tumors, and single-cell RNA-seq analysis inferred their involvement in paracrine crosstalk between epithelial and stromal cells. Collectively, these observations demonstrate that ESR1 mutant tumors gain basal features with induction of basal cytokeratins via epigenetic mechanisms in rare subpopulation of cells. This is associated with increased immune activation, encouraging additional studies of immune therapeutic vulnerabilities in ESR1 mutant tumors.

cancer biology↗

bioRxiv2020

Single cell transcriptomic heterogeneity in invasive ductal and lobular breast cancer cells

Invasive lobular breast carcinoma (ILC), one of the major breast cancer histological subtypes, exhibits unique clinical and molecular features compared to the other well-studied ductal cancer subtype (IDC). The pathognomonic feature of ILC is loss of E-cadherin, mainly caused by inactivating mutations within the CDH1 gene, but the extent of contribution of this genetic alteration to ILC-specific molecular characteristics remains largely understudied. To profile these features transcriptionally, we conducted single cell RNA sequencing on a panel of IDC and ILC cell lines, as well as an IDC cell line (T47D) with CRISPR-Cas9-mediated knock out (KO) of CDH1. Inspection of intra-cell line heterogeneity illustrated genetically and transcriptionally distinct subpopulations in multiple cell lines and highlighted rare populations of MCF7 cells highly expressing an apoptosis-related signature, positively correlated with a pre-adaptation signature to estrogen deprivation. Investigation of CDH1 KO-induced alterations showed transcriptomic membranous systems remodeling, elevated resemblance to ILCs in regulon activation, and suggests IRF1 as a potential mediator of reduced proliferation and increased cytokine-mediated immune-reactivity in ILCs.

cancer biology↗

bioRxiv2019

A luciferase-based assay identifies niclosamide derivatives antagonizing Mcl-1 through post-translational down-regulation

The up-regulation of Mcl-1 expression is a major mechanism of cancer cell survival and therapy resistance. However, the underlying molecular mechanism remains incompletely understood, limiting the number of druggable approaches to selectively inhibit Mcl-1 function. In this study, we designed and employed a novel mechanistic high-throughput screening system to selectively uncover post-translational modulators of Mcl-1. We generated a cell-based high-throughput screening assay in which myeloid leukemia K562 cells constitutively express Mcl-1 or Bcl-xL fused with luciferase (Luc-Mcl-1 or Luc-Bcl-xL, respectively) under a viral promoter. 1,650 bioactive compounds were screened for their ability to selectively induce Mcl-1 down-regulation in a 2-hour assay. A family of niclosamide derivatives were eventually identified for their remarkable ability to decrease Mcl-1 protein stability, exemplified by N007. These salicylate derivatives did not alter Mcl-1 mRNA levels, but selectively induced proteasome-dependent Mcl-1 down-regulation independent of Noxa, Mule, or GSK3{beta}. We also demonstrate that N007 potently induced cell death in leukemia cell lines, including those resistant to Bcl-2 inhibitors. Our work highlights the versatility of the mechanistic high-throughput screening approach as a valuable tool in identifying novel agents with the ability to down-regulate proteins crucial to human diseases.

biochemistry↗

bioRxiv2018

Loss of E-cadherin enhances IGF1-IGF1R pathway activation and sensitizes breast cancers to anti-IGF1R inhibitors

PurposeInsulin-like growth factor I (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin (CDH1) as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R therapies in breast cancer.\n\nExperimental DesignBreast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R therapies. In situ proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data was used to compare IGF1R activation in estrogen receptor positive (ER+) invasive lobular carcinoma (ILC) and invasive ductal carcinoma (IDC) tumors. ER+ ILC cell lines and xenograft tumor explant cultures were used to evaluate efficacy to IGF1R pathway inhibition in combination with endocrine therapy.\n\nResultsDiminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R therapies. PLA demonstrated a direct endogenous interaction between IGF1R and E-cadherin at points of cell-cell contact. Increased expression of IGF1 ligand and levels of IGF1R phosphorylation were observed in E-cadherin deficient ER+ ILC compared to IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R inhibition reduced proliferation in ILC tumor explant culture.\n\nConclusionsWe provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin deficient breast cancers.\n\nSTATEMENT OF SIGNIFICANCEIGF1R signaling is an attractive therapeutic target in breast cancer due to its regulation of proliferation, migration, and invasion. However, clinical trials targeting IGF1R have largely been unsuccessful due to lack of biomarkers to stratify patients for therapeutic response. In this study, we demonstrate loss of E-cadherin as a potential biomarker for response to anti-IGF1R therapy, and show efficacy of IGF1R inhibition in ER+ ILC in combination with endocrine therapy. Patients with ER+ ILC have poorer long-term outcomes than patients with ER+ IDC and have a propensity for increased late recurrences, highlighting the need for improved therapeutic strategies for this subtype of breast cancer. Here, we credential IGF1R inhibition as a novel therapeutic strategy in combination with endocrine therapy for the treatment of ER+ ILC.

cancer biology↗