Increasing matrix stiffness caused by the extracellular matrix (ECM) deposition surrounding cancer cells is accompanied by epithelial–mesenchymal transition (EMT). Here, we show that expression levels of EMT marker genes along with discoidin domain receptor 2 (DDR2) can increase upon matrix stiffening. DDR2 silencing by short hairpin RNA downregulated EMT markers. Promoter analysis and chromatin immunoprecipitation revealed that c-Myb and LEF1 may be responsible for DDR2 induction during cell culture on a stiff matrix. Mechanistically, c-Myb acetylation by p300, which is upregulated on the stiff matrix, seems to be necessary for the c-Myb-and-LEF1–mediated DDR2 expression. Finally, we found that the c-Myb–DDR2 axis is crucial for lung cancer cell line proliferation and expression of EMT marker genes in a stiff environment. Thus, our results suggest that DDR2 regulation by p300 expression and/or c-Myb acetylation upon matrix stiffening may be necessary for regulation of EMT and invasiveness of lung cancer cells.

Discoidin domain receptors (DDRs) are unusual receptor tyrosine kinases (RTKs) that are activated by fibrillar collagens instead of soluble growth factors. DDRs play an important role in various cellular functions and disease processes, including malignant progression. Compared to other RTKs, DDRs have relatively long juxtamembrane domains, which are believed to contribute to receptor function. Despite this possibility, the function and mechanism of the juxtamembrane domain of DDRs have not yet been fully elucidated. In this study, we found that the cytoplasmic juxtamembrane 2 (JM2) region of DDR2 contributed to receptor dimerization, which is critical for receptor activation in response to collagen stimulation. A collagenbinding assay showed that JM2 was required for efficient binding of collagen to the discoidin (DS) domain. Immunohistochemical analysis of DDR2 expression using a tissue microarray demonstrated that DDR2 was overexpressed in several carcinoma tissues, including bladder, testis, lung, kidney, prostate and stomach. In H1299 cells, inhibition of DDR2 activity by overexpressing the juxtamembrane domain containing JM2 suppressed collagen-induced colony formation, cell proliferation and invasion via the inhibition of matrix metalloproteinase-2 and matrix metalloproteinase-9. Taken together, our results suggest that JM2-mediated dimerization is likely to be essential for DDR2 activation and cancer progression. Thus, inhibition of DDR2 function using a JM2-containing peptide might be a useful strategy for the treatment of DDR2-positive cancers.Cell surface receptors sense and respond to diverse extracellular signals, which in turn initiate appropriate cellular responses. Receptor tyrosine kinases (RTKs) constitute a major class of cell surface receptors with high affinities for various polypeptide growth factors, cytokines and hormones. Hence, RTKs have been implicated in normal physiology, and altered or abnormal RTK regulation has been implicated in pathology, including cancer progression. [1][2][3] In the classical model of RTK activation, receptors exist as monomers in the absence of a ligand and dimerize on ligand binding. 4 This dimerization brings the cytoplasmic domains into close proximity, leading to transphosphorylation. 5 However, several studies have shown that receptors for cytokines and growth factors exist in preformed dimeric or trimeric states in the absence of a ligand through their transmembrane (TM) domains or through extracellular juxtamembrane (EJM) and intracellular juxtamembrane (IJM) regions, and these receptors undergo conformational changes in the catalytic domain in response to ligand stimulation. [6][7][8] It was reported that the IJM region of several RTKs contained phosphorylated Tyr residues, which are used as binding sites for proteins involved in downstream signaling, indicating that IJM has a functional role in RTK activation. 9 For example, the IJM of epidermal growth factor receptor (EGFR) has an autoactivating role. 10 However, the precise molecular mechanism has ...

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Dynamic interaction between cancer cells and the surrounding microenvironment is critical for cancer progression via changes in cellular behavior including alteration of secreted molecules. However, the molecular mechanisms underlying the influence exerted by the cancer microenvironment on secretion of molecules during cancer progression remain largely unknown. In this study, we report that secretion of spingsine-1-phosphate (S1P) and its regulator, SphK1 expression is dependent of the substrate rigidity, which is critical for the balance between cancer cell invasion and adhesion. Conditioned media (CM) of MDA-MB-231, an aggressive breast cancer cell obtained from soft substrate (~0.5 kPa) induced chemo-attractive invasion, while CM obtained from stiff substrate (~2.5 kPa) increased cell adhesion instead. We found that the expression of SphK1 is upregulated in the stiff substrate, resulting in an increase in S1P levels in the CM. We also found that upregulation of SphK1 expression in the stiff substrate is dominant in metastatic cancer cells but not in primary cancer cells. These results suggest that alterations in the mechanical environment of the ECM surrounding the tumor cells actively regulate cellular properties such as secretion, which in turn, may contribute to cancer progression.

Microtubules are one of the major targets for anticancer drugs because of their role in cell proliferation and migration. However, as anticancer drugs targeting microtubules have side effects, including the death of normal cells, it is necessary to develop anticancer agents that can target microtubules by specifically acting on cancer cells only. In this study, we identified chemicals that can act as anticancer agents by specifically binding to acetylated microtubules, which are predominant in triple-negative breast cancer (TNBC). The chemical compounds disrupted acetylated microtubule lattices by interfering with microtubule access to alpha-tubulin acetyltransferase 1 (αTAT1), a major acetyltransferase of microtubules, resulting in the increased apoptotic cell death of MDA-MB-231 cells (a TNBC cell line) compared with other cells, such as MCF-10A and MCF-7, which lack microtubule acetylation. Moreover, mouse xenograft experiments showed that treatment with the chemical compounds markedly reduced tumor growth progression. Taken together, the newly identified chemical compounds can be selective for acetylated microtubules and act as potential therapeutic agents against microtubule acetylation enrichment in TNBC.

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Mammalian diaphanous-related formin 1 (mDia1) expression has been linked with progression of malignant cancers in various tissues. However, the precise molecular mechanism underlying mDia1-mediated invasion in cancer cells has not been fully elucidated. In this study, we found that mDia1 is upregulated in invasive breast cancer cells. Knockdown of mDia1 in invasive breast cancer profoundly reduced invasive activity by controlling cellular localization of membrane type 1-matrix metalloproteinase (MT1-MMP) through interaction with microtubule tracks. Gene silencing and ectopic expression of the active form of mDia1 showed that mDia1 plays a key role in the intracellular trafficking of MT1-MMP to the plasma membrane through microtubules. We also demonstrated that highly invasive breast cancer cells possessed invasive activity in a 3D culture system, which was significantly reduced upon silencing mDia1 or MT1-MMP. Furthermore, mDia1-deficient cells cultured in 3D matrix showed impaired expression of the cancer stem cell marker genes, CD44 and CD133. Collectively, our findings suggest that regulation of cellular trafficking and microtubule-mediated localization of MT1-MMP by mDia1 is likely important in breast cancer invasion through the expression of cancer stem cell genes.