ORF24-05 - E-Cadherin Loss Reprograms Metabolic Identities and Drives Tumorigenesis in Human Mammary Epithelial Cells.

Invasive lobular carcinoma (ILC) is a histologic subtype of breast cancer characterized by the genetic loss of CDH1 gene, resulting in loss of the cell adhesion molecule and tumor suppressor E-cadherin. While loss of CDH1 is an early molecular event in ILC, the mechanisms by which it initiates and drives tumorigenesis remain unclear. We hypothesize that loss of E-cadherin alters cell signaling pathways and global gene expression in human mammary epithelial cells (HMECs) to promote ILC tumorigenesis.

To test the effects of E-cadherin loss during ILC tumorigenesis, we established a CRISPR/Cas9 approach to knockout (KO) CDH1 in primary HMECs. 3D Matrigel studies showed larger and more proliferative (Ki67+) acini from CDH1 KO cells, supporting tumorigenesis. We performed RNA-seq from two independent CDH1-targeting gRNAs vs a non-targeting gRNA (NTG) control. Pathway analysis of shared genes suggests enrichment of proliferative pathways, as well as the repression of epithelial-mesenchymal transition and glycolysis in the CDH1 KO sample, consistent with human ILC data. In addition, we performed reverse phase protein array (RPPA) studies to assess changes in cellular signaling in response to CDH1 KO. These data showed increased expression of the glutamine transporter ASCT2 (SLC1A5 gene) and decreased expression of the lactate transporter MCT4, and AMP-activated protein kinase (AMPKα) in the CDH1 KO cells; further highlighting metabolic changes associated with CDH1 loss.

Next, we performed a series of Seahorse assays to determine how CDH1 loss impacts cellular metabolism in HMECs. Seahorse ATP rate assay showed a decrease in glycolysis and an increase in mitochondrial oxidative phosphorylation (OXPHOS) in CDH1 KO cells compared to control. The Mito Flex Fuel Assay indicated similar glucose pathway dependence in both control and CDH1 KO cells, but CDH1 KO cells showed increased glutamine pathway dependence, suggesting compensatory mitochondrial oxidation of glutamine in the CDH1 KO cells. Mito Stress test assays showed that CDH1 KO cells have an increased spare respiratory capacity, which further decreased in the absence of glutamine.

Based on these observations, we screened various metabolic inhibitors. We found that metformin and the SLC1A5 inhibitor (V9302) both inhibited proliferation of CDH1 KO cells in 2D and 3D Matrigel cultures, decreasing acini size and percentage of Ki67+ acini. The Mito Stress test with these inhibitors also showed a decrease in the percent spare respiratory capacity in the CDH1 KO cells, suggesting inhibition of mitochondrial respiration and OXPHOS. Understanding how E-cadherin loss reprograms early metabolic events in ILC could have potential translational impacts and lead to novel therapeutic approaches for prevention and treatment of ILC.