Knockout model reveals the role of Nischarin in mammary gland development, breast tumorigenesis and response to metformin treatment

Abstract

Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7–10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus‐polyoma middle T‐antigen (MMTV‐PyMT) oncogene. The MMTV‐PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild‐type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin‐deficient breast cancers.

Nischarin Deletion Reduces Oxidative Metabolism and Overall ATP: A Study Using a Novel NISCHΔ5-6 Knockout Mouse Model

“…Our findings show that Nisch can also influence mesenchymal cells, which supports the idea that Nisch facilitates neuronal development [2]. Previous studies also address Nisch's role in the embryological development of the gut and reproductive organs [10].…”

“…Consistent with Nisch-mutated mice used in previous studies, Nisch ∆5-6 mice display a smaller size and attenuated growth phenotype [8,10,11]. From the first day of birth, these mice are observed to be smaller than the wild-type and heterozygous mice, which are indistinguishable from each other.…”

“…The only other time the Seahorse assay was performed to study Nisch was in the lossof-function Nisch-mutated mice that have been crossed with the PyMT oncogene, which causes spontaneous mammary tumors in mice [10]. Mice were bred to obtain Nisch −/− -PyMT and Nisch +/+ -PyMT mice.…”

“…A schematic of possible strategies is represented in Figure 1a. Nisch-tm1b mice were backcrossed onto the C57BL/6J background, in order to compare our results with previous studies [10]. Genomic DNA PCR was performed.…”

“…These exons do not harbor any motifs that suggest protein function; thus, we predict them to be involved in mRNA or protein stabilization. Currently, there are two mouse models for Nisch: Zhang et al generated a knockout model through the deletion of exon 4, and Dong et al generated a loss-of-function model through the deletion of exons 7-10 [10,11]. Using the model proposed in this paper, we investigated migration and metabolism in cells without the context of cancer.…”