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The Role of Runx2 in Microtubule Acetylation in Bone Metastatic Breast Cancer Cells

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Breast cancer is the most commonly diagnosed cancer type, making up a quarter of all cases among women. While modern-day research has shown tremendous progress in the development of treatment options, complications related to bone metastasis remain an obstacle that demands further attention. Cancer cells that migrate into the bone microenvironment show significant alterations in their metabolism and gene expression profiles. In this study, we set out to better understand the changes induced by Runx2 in bone-derived breast cancer cells. We identified key changes in the stability of the microtubule cytoskeleton of bone-derived cells expressing Runx2. These changes have implications on a variety of processes related to metabolism, cellular stress response, and response to common chemotherapies. These studies help to shed light on the changes that take place as a tumor begins to metastasize and to better predict which therapies will benefit patients with bone metastasis.

Abstract

Bone metastasis of breast cancer results in severe bone loss, fractures, and death. Crosstalk between breast cancer cells and bone resident cells promotes osteoclast activity and the release of growth factors from the bone matrix resulting in aggressive tumor growth and bone loss. We and others have shown that Runt-related transcription factor-2 (Runx2) promotes metastatic tumor growth-associated bone loss. Breast cancer cells also induce autophagy to survive metabolic stress at the metastatic site. Recently, we reported a Runx2-dependent increase in autophagy. In this study, to examine the underlying mechanisms of metastasis and tumor resistance to stress, we used a bone metastatic isogenic variant of breast cancer MDA-MB-231 cells isolated from a xenograft tumor mouse model of metastasis. Our results with immunofluorescence and biochemical approaches revealed that Runx2 promotes microtubule (MT) stability to facilitate autophagy. Stable MTs are critical for autophagosome trafficking and display increased acetylation at Lysine 40 of α-tubulin. Runx2 silencing decreases acetylated α-tubulin levels. The expression levels of HDAC6 and αTAT1, which serve to regulate the acetylation of α-tubulin, were not altered with Runx2 silencing. We found that HDAC6 interaction with α-tubulin is inhibited by Runt-related factor-2 (Runx2). We show that the expression of wild-type Runx2 can restore the acetylated polymer of MTs in Runx2 knockdown cells, while the C-terminal deletion mutant fails to rescue the polymer of MTs. Importantly, cellular stress, such as glucose starvation also increases the acetylation of α-tubulin. We found that the loss of Runx2 increases the sensitivity of breast cancer cells to MT-targeting agents. Overall, our results indicate a novel regulatory mechanism of microtubule acetylation and suggest that Runx2 and acetylated microtubules may serve as therapeutic targets for bone metastatic tumors.

SUBMITTER: Othman A

PROVIDER: S-EPMC9323014 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The majority of the deaths from breast cancer is due to metastasis. Bone is the most common organ to which breast cancer cells metastasize. The mechanism regulating the bone-metastatic preference remains unclear; there is a lack of a gene signature to distinguish bone-metastatic breast cancer cells. Herein, florescence-labeled MDA-MB-231 cells were transplanted into the fat pads of of the mammary gland in nude mice to generate breast tumors. Tumor cells invaded into the circulation were tracked by in vivo flow cytometry system. Metastatic tumor cells in the bone were isolated using fluorescent-activated cell sorting technique, followed by assays of cell colony formation, migration and invasion, mammosphere formation in vitro, mammary gland tumorigenesis in vivo, and Next-Generation Sequencing analysis as well. Through tumor regeneration and cell sorting, two bone-metastatic cell sublines were derived from MDA-MB-231 cells; which showed higher abilities to proliferate, migrate, invade and epithelial-to-mesenchymal transit in vitro, and stronger ability to regenerate tumors and metastasize to the bone in vivo. Both cell sublines exhibited cancer stem cell-like characteristics including higher expression levels of stem cell markers and stronger ability for mommaspheres formation. Furthermore, a Normal Distribution-like pattern of the bone-metastatic cells invading into circulation was firstly identified. Deep-sequencing analysis indicated upregulation of multiple signaling pathways in regulating EMT, cell membrane budding and morphologic change, lipid metabolism, and protein translation, which are required to provide adequate metabolic enzymes, structural proteins, and energy for the cells undergoing metastasis. In conclusion, we established two bone-metastatic breast cancer cell sublines, carrying higher degree of stemness and malignancy. The gene signature distinguishing the bone-metastatic breast cancer cells holds therapeutic potentials in prevention of breast cancer metastasis to the bone.

Project description:IntroductionBreast cancers frequently metastasise to the skeleton where they cause osteolytic bone destruction by stimulating osteoclasts to resorb bone and by preventing osteoblasts from producing new bone. The Runt-related transcription factor 2, Runx2, is an important determinant of bone metastasis in breast cancer. Runx2 is known to mediate activation of osteoclast activity and inhibition of osteoblast differentiation by metastatic breast cancer cells. However, while Runx2-regulated genes that mediate osteoclast activation have been identified, how Runx2 determines inhibition of osteoblasts is unknown.MethodsThe aim of this study was to determine how Runx2 mediates the ability of metastatic breast cancer cells to modulate the activity of bone cells. We have previously demonstrated that Runx2 requires the co-activator core binding factor beta (CBFβ) to regulate gene expression in breast cancer cells. We, therefore, performed independent microarray analyses to identify target genes whose expression is dependent upon both Runx2 and CBFβ. Common target genes, with a role in modulating bone-cell function, were confirmed using a combination of siRNA, quantitative reverse transcriptase PCR (qRT-PCR), ELISA, promoter reporter analysis, Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation (ChIP) assays. The function of Runx2/CBFβ-regulated genes in mediating the ability of MDA-MB-231 to inhibit osteoblast differentiation was subsequently established in primary bone marrow stromal cell cultures and MC-3T3 osteoblast cells.ResultsWe show that Runx2/CBFβ mediates inhibition of osteoblast differentiation by MDA-MB-231 cells through induction of the Wnt signaling antagonist, sclerostin. We demonstrate that MDA-MB-231 cells secrete sclerostin and that sclerostin-expression is critically dependent on both Runx2 and CBFβ. We also identified the osteoclast activators IL-11 and granulocyte-macrophage colony-stimulating factor (GM-CSF) as new target genes of Runx2/CBFβ in metastatic breast cancer cells.ConclusionsThis study demonstrates that Runx2 and CBFβ are required for the expression of genes that mediate the ability of metastatic breast cancer cells to directly modulate both osteoclast and osteoblast function. We also show that Runx2-dependent inhibition of osteoblast differentiation by breast cancer cells is mediated through the Wnt antagonist, sclerostin.

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The Role of Runx2 in Microtubule Acetylation in Bone Metastatic Breast Cancer Cells

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