Impaired intranuclear trafficking of Runx2 (AML3/CBFA1) transcription factors in breast cancer cells inhibits osteolysis in vivo

Abstract

Runx transcription factors comprise a family of proteins that are essential for organogenesis. A unique nuclear matrix-targeting signal in the C terminus directs these factors to their appropriate subnuclear domains. At these sites, they interact with coregulatory proteins and target genes. We have previously shown that aberrant expression of the Runx2 DNA binding domain in metastatic breast cancer cells can prevent production of osteolytic lesions in bone. Here, we show that proper Runx2 subnuclear targeting is required for osteolysis. We have identified point mutations of the Runx2 nuclear matrix-targeting signal sequence that impair its targeting to nuclear matrix sites. These mutations block the invasive and osteolytic properties of MDA-MB-231 breast cancer cells in vivo. Cell lines expressing this Runx2 mutant protein inhibit the osteogenic properties of bone marrow stromal cells in coculture assays. The mutant breast cancer cells also exhibit reduced invasiveness in vitro and do not express genes involved in invasion and angiogenesis (VEGF and MMP13). Our findings suggest that fidelity of Runx2 intranuclear organization is necessary for expression of target genes that mediate the osteolytic activity of metastatic breast cancer cells.gene expression ͉ intranuclear organization ͉ nuclear matrix R unx (AML͞CBFA͞PEBP2) transcription factors are tissuespecific regulatory proteins that are involved in the control of hematopoiesis (Runx1͞AML1), osteogenesis (Runx2͞ AML3), and neural and gastrointestinal cell differentiation (Runx3͞AML2) (1-3). Translocations or mutations of Runx1 results in leukemogenesis (4), whereas mutations of Runx2 and Runx3 lead to skeletal disease (5) and stomach cancer (3), respectively. The Runx transcription factors function as scaffolds for interaction with various coregulatory proteins. These complexes interact in a combinatorial fashion to regulate gene expression. Furthermore, Runx proteins are focally localized within the nucleus. The subnuclear localization of Runx proteins requires at least two trafficking signals; one signal supports nuclear import (nuclear localization signals) and the other signal is required for residency in nuclear matrix-associated regulatory domains (nuclear matrix-targeting signal; NMTS) (6-9). The role of Runx proteins in both gene expression and nuclear structure is a reflection of their modular organization. The N terminus binds specific DNA sequences, whereas the C-terminal domain interacts with coregulatory factors and associates with the nuclear matrix (10-13). During tissue differentiation, Runx proteins form stage-specific complexes with subsets of coactivators (14-16), corepressors (17-19), and factors that are end points of key signaling cascades (20,21). These multimeric complexes are organized in punctuate subnuclear foci that constitute nuclear microenvironments (22).The critical role of Runx2 in osteogenesis has provided a model for investigating the importance of subnuclear localization for tissue differentiation. Mice homozygous for Runx2 ...

A role for Runx2 in normal mammary gland and breast cancer bone metastasis

“…The effect of perturbing Runx2 nuclear matrix targeting on osteogenic differentiation was similar to that observed with the dominantnegative Runx2D230 protein [Barnes et al, 2004]. Importantly, the mutant Runx2 protein also inhibited the invasive and osteolytic properties of MDA-MB-231 cells as determined by their reduced migration ability on Matrigel and a reduction in the formation of osteolytic lesions in vivo [Javed et al, 2005]. These findings correlated with reduced expression of VEGF and collagenase-3, both factors known to be associated with cell invasion.…”

“…The correct localization of Runx2 to intranuclear subdomains has also been shown to be important for expression of target genes required for the osteolytic activity of metastatic breast cancer cells [Javed et al, 2005]. Runx2 contains a nuclear matrix-targeting signal (NMTS) in the C-terminal region which is responsible for its association with the nuclear matrix.…”

Breast cancer bone metastasis mediated by the Smad tumor suppressor pathway

“…This TGF-␤ responsive element is recognized by Smad2͞3-Smad4 in complex with RUNX family members and responds to TGF-␤ in many different cell lines (24,25). RUNX activity in breast cancer cells is implicated in osteolytic bone metastasis (26).…”

Transcription factor Runx2 is a regulator of epithelial–mesenchymal transition and invasion in thyroid carcinomas

“…8,20 In breast carcinoma cells, expression of Runx2 is shown to be important in the positive regulation of angiogenic factors, and Runx2-regulated MMPs are functionally related to the invasion properties of breast cancer cells. 29,30 Likewise, our current study demonstrated that Runx2 expression is involved with positive regulation of angiogenic factors (VEGFA, VEGFC), MMP2 and EMTrelated molecules (SNAI2, SNAI3, TWIST1) in thyroid carcinoma. Furthermore, we showed that Runx2 silencing repressed the invasion of thyroid carcinoma cells in the transwell cell invasion assay.…”