Cadmium Exposure Inhibits Branching Morphogenesis and Causes Alterations Consistent With HIF-1α Inhibition in Human Primary Breast Organoids

Abstract

Developmental cadmium exposure in vivo disrupts mammary gland differentiation, while exposure of breast cell lines to cadmium causes invasion consistent with the epithelial-mesenchymal transition (EMT). The effects of cadmium on normal human breast stem cells have not been measured. Here, we quantified the effects of cadmium exposure on reduction mammoplasty patient-derived breast stem cell proliferation and differentiation. Using the mammosphere assay and organoid formation in 3D hydrogels, we tested 2 physiologically relevant doses of cadmium, 0.25 and 2.5 µM, and tested for molecular alterations using RNA-seq. We functionally validated our RNA-seq findings with a hypoxia-inducible factor (HIF)-1α activity reporter line and pharmaceutical inhibition of HIF-1α in organoid formation assays. 2.5 µM cadmium reduced primary mammosphere formation and branching structure organoid formation rates by 33% and 87%, respectively. Despite no changes in mammosphere formation, 0.25 µM cadmium inhibited branching organoid formation in hydrogels by 73%. RNA-seq revealed cadmium downregulated genes associated with extracellular matrix formation and EMT, while upregulating genes associated with metal response including metallothioneins and zinc transporters. In the RNA-seq data, cadmium downregulated HIF-1α target genes including LOXL2, ZEB1, and VIM. Cadmium significantly inhibited HIF-1α activity in a luciferase assay, and the HIF-1α inhibitor acriflavine ablated mammosphere and organoid formation. These findings show that cadmium, at doses relevant to human exposure, inhibited human mammary stem cell proliferation and differentiation, potentially through disruption of HIF-1α activity.

Roles of Lysine-63-linked ubiquitination on Hif-1α in cell fate decisions between cell proliferation and apoptosis under cadmium effects in epithelial cells

“…However, some of the effects of Cd impair CA-XI production under hypoxic conditions. This indicated that Cd prevents HIF-1α protein from stimulating CA-XI expression in response to hypoxic conditions or decreases HIF-1binding activity under normoxic conditions [49,50]. Unlike our study, the last works did not show the mechanism by which Cd stabilized Hif-1α, but they noted that Cd inhibited proliferation and differentiation through disruption of HIF-1α activity under normoxic conditions.…”

Advances in Hydrogels in Organoids and Organs‐on‐a‐Chip

“…The development of the intestinal organoid system in 2009 was a major technological advance for the stem cell organoids field. Since then, an extensive body of works have improved the methodology and expanded the range of tissue and organ types that can be investigated, such as brain, gastric intestine, liver, kidney, lung, pancreas, prostate, retina, cardic, blood vessel, and mammary gland . Their versatile nature and potential to generate human tissues endow organoids with a variety of applications.…”

Applying Cell Painting in Non-Tumorigenic Breast Cells to Understand Impacts of Common Chemical Exposures

“…The role of hypoxia and inhibition of EGLN1, a hypoxia-inducible factor prolyl hydroxylase, by FG-4592 has been described earlier in both pancreatic and ovarian cancer (Fujimoto et al 2019, Price et al 2019). The role of HIF-1A being inhibited by cadmium has been observed in human primary breast organoids, where we identified that HIF-1A was essential for mammary organoid formation (Rocco et al 2018). Characterizing how heavy metals interact with hypoxia signaling will be an important area of future research to understand potential mechanis t ic links between breast development and breast cancer.…”