BackgroundEstrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer.Patients and methodsTo identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287–395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots.ResultsWe identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3′ partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations.ConclusionsCollectively, these data indicate that N-terminal ESR1 fusions involving exons 6–7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

Insulin-like growth factor 1 (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin () as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R/insulin receptor (InsR) therapies in breast cancer. Breast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R/InsR therapies. proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data were used to compare IGF1R/InsR 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. Diminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R/InsR 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/InsR phosphorylation were observed in E-cadherin-deficient ER+ ILC compared with IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R/InsR inhibition reduced proliferation in ILC tumor explant culture. We provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R/InsR targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin-deficient breast cancers. .

Insulin and insulin-like growth factor I (IGF1) influence cancer risk and progression through poorly understood mechanisms. To better understand the roles of insulin and IGF1 signaling in breast cancer, we combined proteomic screening with computational network inference to uncover differences in IGF1 and insulin induced signaling. Using reverse phase protein array, we measured the levels of 134 proteins in 21 breast cancer cell lines stimulated with IGF1 or insulin for up to 48 h. We then constructed directed protein expression networks using three separate methods: (i) lasso regression, (ii) conventional matrix inversion, and (iii) entropy maximization. These networks, named here as the time translation models, were analyzed and the inferred interactions were ranked by differential magnitude to identify pathway differences. Insulin (Ins)1 and type I insulin-like growth factor (IGF1) receptors (InsR and IGF1R, respectively) are receptor tyrosine kinases that are expressed in almost all types of cells. Signaling through InsR and IGF1R initiates a phosphorylation cascade that drives cell growth and proliferation (1-8). Overexpression of these receptors is correlated with higher breast cancer risk (9 -15) and has been shown to influence tumorigenesis, metastasis, and resistance to existing forms of cancer therapy (10,16,17). IGF receptor blockade can slow tumor growth and metastasis, but the receptor has proven to be difficult to target specifically (18 -20). A confounding factor in developing therapies targeting these receptors is their high sequence (ϳ60%) and structural homology. IGF1R and InsR are able to form functional hybrids, and each can partially compensate for the loss or suppression of the other (1-4, 21-24). Moreover, it has been shown that IGF1R signaling is one mechanism of resistance to conventional hormonal therapy (19,(25)(26)(27)(28)(29). Understanding the relationships between IGF1R and InsR signaling cascades and their combinatorial role in cancer is crucial to developing better diagnostics and personalized treatments for cancer.

No abstract

Breast cancer development and progression is

Increased AMP-activated protein kinase (AMPK) activity leads to enhanced fatty acid utilization, while also promoting increased ubiquitin-dependent proteolysis (UDP) in mammalian skeletal muscle. β-guanidinopropionic acid (βGPA) is a commercially available dietary supplement that has been shown to promote an AMPK-dependent increase in fatty acid utilization and aerobic capacity in mammals by compromising creatine kinase function. However, it remains unknown if continuous βGPA supplementation can negatively impact skeletal muscle growth in a rapidly growing juvenile. The current study was conducted to examine the effect of βGPA supplementation on whole-body and skeletal muscle growth in juvenile and young adult mice. Three-week old, post weanling CD-1 mice were fed a standard rodent chow that was supplemented with either 2 % (w/w) α-cellulose (control) or βGPA. Control and βGPA-fed mice (n = 6) were sampled after 2, 4, and 8 weeks. Whole-body and hindlimb muscle masses were significantly (P < 0.05) reduced in βGPA-fed mice by 2 weeks. The level of AMPK (T172) phosphorylation increased significantly (P < 0.05) in the gastrocnemius of βGPA-fed versus control mice at 2 weeks, but was not significantly different at the 4- and 8-week time points. Further analysis revealed a significant (P < 0.05) increase in the skeletal muscle-specific ubiquitin ligase MAFbx/Atrogin-1 protein and total protein ubiquitination in the gastrocnemius of βGPA versus control mice at the 8-week time point. Our data indicate that feeding juvenile mice a βGPA-supplemented diet significantly reduced whole-body and skeletal muscle growth that was due, at least in part, to an AMPK-independent increase in UDP.

<div>Abstract<p><b>Purpose:</b> Insulin-like growth factor 1 (IGF1) signaling regulates breast cancer initiation and progression and associated cancer phenotypes. We previously identified E-cadherin (<i>CDH1</i>) as a repressor of IGF1 signaling and in this study examined how loss of E-cadherin affects IGF1R signaling and response to anti-IGF1R/insulin receptor (InsR) therapies in breast cancer.</p><p><b>Experimental Design:</b> Breast cancer cell lines were used to assess how altered E-cadherin levels regulate IGF1R signaling and response to two anti-IGF1R/InsR therapies. <i>In situ</i> proximity ligation assay (PLA) was used to define interaction between IGF1R and E-cadherin. TCGA RNA-seq and RPPA data were used to compare IGF1R/InsR 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.</p><p><b>Results:</b> Diminished functional E-cadherin increased both activation of IGF1R signaling and efficacy to anti-IGF1R/InsR 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/InsR phosphorylation were observed in E-cadherin–deficient ER+ ILC compared with IDC tumors. IGF1R pathway inhibitors were effective in inhibiting growth in ER+ ILC cell lines and synergized with endocrine therapy and similarly IGF1R/InsR inhibition reduced proliferation in ILC tumor explant culture.</p><p><b>Conclusions:</b> We provide evidence that loss of E-cadherin hyperactivates the IGF1R pathway and increases sensitivity to IGF1R/InsR targeted therapy, thus identifying the IGF1R pathway as a potential novel target in E-cadherin–deficient breast cancers. <i>Clin Cancer Res; 24(20); 5165–77. ©2018 AACR</i>.</p></div>

<p>Supplementary Figure / Table Legends</p>