The transcription factor Runx2 is highly expressed in breast cancer cells compared with mammary epithelial cells and contributes to metastasis. Here we directly show that Runx2 expression promotes a tumor cell phenotype of mammary acini in three-dimensional culture. Human mammary epithelial cells (MCF-10A) form polarized, growth-arrested, acinilike structures with glandular architecture. The ectopic expression of Runx2 disrupts acini formation, and electron microscopic ultrastructural analysis revealed the absence of lumens. Characterization of the disrupted acini structures showed increased cell proliferation (Ki-67 positive cells), decreased apoptosis (Bcl-2 induction), and loss of basement membrane formation (absence of B 4 integrin expression). In complementary experiments, inhibition of Runx2 function in metastatic MDA-MB-231 breast cancer cells by stable expression of either short hairpin RNA-Runx2 or a mutant Runx2 deficient in subnuclear targeting resulted in reversion of acini to more normal structures and reduced tumor growth in vivo. These novel findings provide direct mechanistic evidence for the biological activity of Runx2, dependent on its subnuclear localization, in promoting early events of breast cancer progression and suggest a molecular therapeutic target.

Changes in nuclear morphology occur during normal development and have been observed during the progression of several diseases. The shape of a nucleus is governed by the balance of forces exerted by nuclear-cytoskeletal contacts and internal forces created by the structure of the chromatin and nuclear envelope. However, factors that regulate the balance of these forces and determine nuclear shape are poorly understood. The SWI/SNF chromatin remodeling enzyme ATPase, BRG1, has been shown to contribute to the regulation of overall cell size and shape. Here we document that immortalized mammary epithelial cells show BRG1-dependent nuclear shape changes. Specifically, knockdown of BRG1 induced grooves in the nuclear periphery that could be documented by cytological and ultrastructural methods. To test the hypothesis that the observed changes in nuclear morphology resulted from altered tension exerted by the cytoskeleton, we disrupted the major cytoskeletal networks and quantified the frequency of BRG1-dependent changes in nuclear morphology. The results demonstrated that disruption of cytoskeletal networks did not change the frequency of BRG1-induced nuclear shape changes. These findings suggest that BRG1 mediates control of nuclear shape by internal nuclear mechanisms that likely control chromatin dynamics.

The ATPase subunits of the SWI/SNF chromatin remodeling enzymes, Brahma (BRM) and Brahma related gene 1 (BRG1), can induce cell cycle arrest in BRM and BRG1 deficient tumor cell lines, and mice heterozygous for Brg1 are predisposed to breast tumors, implicating loss of BRG1 as a mechanism for unregulated cell proliferation. To test the hypothesis that loss of BRG1 can contribute to breast cancer, we utilized RNA interference to reduce the amounts of BRM or BRG1 protein in the nonmalignant mammary epithelial cell line, MCF-10A. When grown in reconstituted basement membrane (rBM), these cells develop into acini that resemble the lobes of normal breast tissue. Contrary to expectations, knockdown of either BRM or BRG1 resulted in an inhibition of cell proliferation in monolayer cultures that was enhanced in three-dimensional rBM culture. This inhibition was strikingly enhanced in three-dimensional rBM culture, although some BRM depleted cells were later able to resume proliferation. Cells did not arrest in any specific stage of the cell cycle; instead, the cell cycle length increased by approximately 50%. Thus, SWI/SNF ATPases promote cell cycle progression in nonmalignant mammary epithelial cells.

Integrin expression and activity are altered in tumors, and aberrant integrin signaling promotes malignancy. However, how integrins become altered in tumors remains poorly understood. We discovered that oncogenic activation of MEK signaling induces cell growth and survival, and promotes the malignant phenotype of mammary epithelial cells (MECs) by increasing α5 integrin expression. We determined that MEK activates c-Myc to reduce the transcription of the SWI/SNF chromatin remodeling enzyme Brahma (BRM). Our studies revealed that reduced BRM expression and/or activity drives the malignant behavior of MECs by epigenetically promoting C/EBPβ expression to directly induce α5 integrin transcription. Consistently, we could show that restoring BRM levels normalized the malignant behavior of transformed MECs in culture and in vivo by preventing C/EBPβ-dependent α5 integrin transcription. Our findings identify a novel mechanism whereby oncogenic signaling promotes malignant transformation by regulating transcription of a key chromatin remodeling molecule that regulates integrin-dependent stromal–epithelial interactions.

No abstract