Parathyroid Hormone-Related Protein Negatively Regulates Tumor Cell Dormancy Genes in a PTHR1/Cyclic AMP-Independent Manner.

Rachelle W Johnson,Patricia W M Ho,T John Martin,Jasmine A Johnson,Nathan J Pavlos,Natalie A Sims,Audrey S M Chan,Yao Sun

doi:10.3389/fendo.2018.00241

Abstract

Parathyroid hormone-related protein (PTHrP) expression in breast cancer is enriched in bone metastases compared to primary tumors. Human MCF7 breast cancer cells “home” to the bones of immune deficient mice following intracardiac inoculation, but do not grow well and stain negatively for Ki67, thus serving as a model of breast cancer dormancy in vivo. We have previously shown that PTHrP overexpression in MCF7 cells overcomes this dormant phenotype, causing them to grow as osteolytic deposits, and that PTHrP-overexpressing MCF7 cells showed significantly lower expression of genes associated with dormancy compared to vector controls. Since early work showed a lack of cyclic AMP (cAMP) response to parathyroid hormone (PTH) in MCF7 cells, and cAMP is activated by PTH/PTHrP receptor (PTHR1) signaling, we hypothesized that the effects of PTHrP on dormancy in MCF7 cells occur through non-canonical (i.e., PTHR1/cAMP-independent) signaling. The data presented here demonstrate the lack of cAMP response in MCF7 cells to full length PTHrP(1–141) and PTH(1–34) in a wide range of doses, while maintaining a response to three known activators of adenylyl cyclase: calcitonin, prostaglandin E2 (PGE2), and forskolin. PTHR1 mRNA was detectable in MCF7 cells and was found in eight other human breast and murine mammary carcinoma cell lines. Although PTHrP overexpression in MCF7 cells changed expression levels of many genes, RNAseq analysis revealed that PTHR1 was unaltered, and only 2/32 previous PTHR1/cAMP responsive genes were significantly upregulated. Instead, PTHrP overexpression in MCF7 cells resulted in significant enrichment of the calcium signaling pathway. We conclude that PTHR1 in MCF7 breast cancer cells is not functionally linked to activation of the cAMP pathway. Gene expression responses to PTHrP overexpression must, therefore, result from autocrine or intracrine actions of PTHrP independent of PTHR1, through signals emanating from other domains within the PTHrP molecule.

Highlights

Parathyroid hormone-related protein (PTHrP, gene name PTHLH/Pthlh) is a cytokine with functions in both pathology and physiology [1, 2]
This work provides extensive evidence that PTHrP, it is capable of inducing substantial changes in gene expression and behavior in MCF7 cells, does not signal through the PTHR1 to activate the cyclic AMP (cAMP) pathway in these cells
PTHR1 is detected by qPCR, no cAMP response was detected, and no activity was observed in a cAMP responsive element binding protein (CREB) reporter assay

Summary

Introduction

Parathyroid hormone-related protein (PTHrP, gene name PTHLH/Pthlh) is a cytokine with functions in both pathology and physiology [1, 2]. Upon ligand binding to the receptor, cyclic AMP (cAMP) is activated, followed by protein kinase A (PKA) activation, cAMP responsive element binding protein (CREB) phosphorylation, and transcription of CREB target genes [10,11,12,13] This PTHR1-dependent signaling pathway is shared between PTH and PTHrP due to high sequence homology in their amino-terminal domains; the portion of the molecule that interacts with the receptor [14]. MCF7 cells possess specific, high affinity receptors for calcitonin linked to adenylyl cyclase activation, and activate cAMP in response to prostaglandin E2 [15, 16] These data suggest that the effect of PTHrP overexpression on tumor dormancy in MCF7 cells may occur through PTHR1-independent actions of the PTHrP molecule

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Results

Conclusion