Loss of E-cadherin Activates a Targetable IGF1R Pathway in Invasive Lobular Breast Carcinoma

Invasive Ductal Carcinoma (IDC) and Invasive Lobular Carcinoma (ILC) are two major subtypes of breast cancer with significant differences in their histological and molecular underpinnings. ILC has a unique hallmark of loss of E-cadherin (CDH1) in 90% of the cases, which we have previously demonstrated as a negative regulator of Insulin-like Growth Factor 1 (IGF1) receptor, IGF1R through a comprehensive analysis of cell line models and tumor samples on TCGA. We propose that the loss of E-cadherin in ILC sensitizes cells to growth factor signaling and thus alters their susceptibility to growth factor signaling inhibition. We generated CDH1 knockout (KO) IDC cell lines to investigate the mechanism by which E-cadherin loss activates IGF pathway and its subsequent effectors while also assessing its targetability in patients. CDH1 KO cells exhibited anchorage independent growth in suspension culture and altered p120 catenin localization as seen in ILC cells. Through in vitro studies, we observed increased signaling sensitivity to IGF/ insulin ligands where the high activation levels are sustained for an extended duration in the KO cells. In addition, there was higher migratory potential in the CDH1 KO cells, which was further enhanced as a chemotactic response to IGF1 or serum and as a haptotactic response to Collagen I. This phenotype was reversed with an IGF1R inhibitor to exhibit phenotype specificity. Despite no consistent differences in membranous IGF1R levels, higher ligand-receptor interaction was observed with E-cadherin loss. Our results currently demonstrate IGF1R’s increased availability for ligand binding which in turn allows for an enhanced signaling activation. As an extension to the pathway activation, increased susceptibility to IGF1R, PI3K, AKT and MEK inhibitors was also observed in T47D CDH1 KO cells. With about 90% of ILC cases being ER+, we investigated and showed the additive effect of Fulvestrant with Akt inhibitors in KO cells. Although a clear susceptibility to Akt inhibitor was not seen in ILC patient-derived organoids (PDO), a favorable trend was observed when compared to IDC PDOs. Our findings elucidated IGF1 signaling repression by E-cadherin in ILC, thus supporting the use of E-cadherin loss as a stratification method for improved targeted therapy approaches

Role of psr-1 C-terminal Domain in Mediating Apoptotic Cell Clearance

Programmed cell death plays a major physiological role in cell elimination in order to maintain cell homeostasis. Cell engulfment and clearance are important final steps of programmed cell death. Apoptotic cell clearance in C.elegans is triggered by alteration in phospholipid asymmetry. Phosphatidylserine (PS), which exists in the inner leaflet of plasma membrane in dormant cells, is externalized onto the outer leaflet during apoptosis and signals phagocytic cells for engulfment. PSR-1(PS Receptor) in C.elegans has been shown as a protein that preferentially binds to PS. PSR-1 was first discovered as a transmembrane protein on phagocytes that engulf apoptotic cells; with the N-terminal region in the cytosol and the C-terminal region in the extracellular space. This implies that the primary function of detecting externalized PS might be performed by the C-terminal portion of the protein. Therefore, single and double mutants of psr-1 C-terminal domain were generated with ced-1, ced-8 and acced-8(sm351). The C-terminal region of psr-1 was found to play an important role in maintaining a regulated cell clearance mechanism. Both versions of psr-1 C-terminal region mutants exhibited an increased cell clearance phenotype. Depending on the length of deletion at C-terminus, the two psr-1 mutants showed different numbers of somatic cell corpse in each embryonic stage when ced-8 was activated (acCED-8).While acced-8(sm351) mutant alone exhibited N2-like phenotype, double mutants of psr-1 with acced-8(sm351) exhibited significantly different phenotype when compared to one another. This observation strongly indicates that PSR-1 C-terminal region is critical for cell corpse clearance in coordination with acCED-8

Proteomic and transcriptomic profiling identifies mediators of anchorage-independent growth and roles of inhibitor of differentiation proteins in invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is a histological subtype of breast cancer with distinct molecular and clinical features from the more common subtype invasive ductal carcinoma (IDC). ILC cells exhibit anchorage-independent growth in ultra-low attachment (ULA) suspension cultures, which is largely attributed to the loss of E-cadherin. In addition to anoikis resistance, herein we show that human ILC cell lines exhibit enhanced cell proliferation in ULA cultures as compared to IDC cells. Proteomic comparison of ILC and IDC cell lines identified induction of PI3K/Akt and p90-RSK pathways specifically in ULA culture in ILC cells. Further transcriptional profiling uncovered unique upregulation of the inhibitors of differentiation family transcription factors ID1 and ID3 in ILC ULA culture, the knockdown of which diminished the anchorage-independent growth of ILC cell lines through cell cycle arrest. We find that ID1 and ID3 expression is higher in human ILC tumors as compared to IDC, correlated with worse prognosis uniquely in patients with ILC and associated with upregulation of angiogenesis and matrisome-related genes. Altogether, our comprehensive study of anchorage independence in human ILC cell lines provides mechanistic insights and clinical implications for metastatic dissemination of ILC and implicates ID1 and ID3 as novel drivers and therapeutic targets for lobular breast cancer

Mapping molecular subtype specific alterations in breast cancer brain metastases identifies clinically relevant vulnerabilities

The molecular events and transcriptional plasticity driving brain metastasis in clinically relevant breast tumor subtypes has not been determined. Here we comprehensively dissect genomic, transcriptomic and clinical data in patient-matched longitudinal tumor samples, and unravel distinct transcriptional programs enriched in brain metastasis. We report on subtype specific hub genes and functional processes, central to disease-affected networks in brain metastasis. Importantly, in luminal brain metastases we identify homologous recombination deficiency operative in transcriptomic and genomic data with recurrent breast mutational signatures A, F and K, associated with mismatch repair defects, TP53 mutations and homologous recombination deficiency (HRD) respectively. Utilizing PARP inhibition in patient-derived brain metastatic tumor explants we functionally validate HRD as a key vulnerability. Here, we demonstrate a functionally relevant HRD evident at genomic and transcriptomic levels pointing to genomic instability in breast cancer brain metastasis which is of potential translational significance