Breast cancer is the most common type of cancer among women worldwide, and metastasis represents the most devastating stage of the disease. Recent studies have revealed that microRNAs (miRNA) have critical roles to regulate cancer cell invasion and metastasis. Here we present evidence to show the role of miR-182 in breast cancer metastasis. miR-182 is upregulated in the malignant cell line variants of both human MCF10 and mouse 4T1 series. Ectopic expression of miR-182 enhanced breast cancer cell motility and invasiveness, whereas miR-182 inhibition resulted in opposite changes. In nude mice, miR-182 led to increased pulmonary colonization of cancer cells. We further demonstrated that miR-182 directly targets MIM (Missing in Metastasis), which suppresses metastasis by inhibiting ras homolog family member A (RhoA) activity and stress fiber formation in breast cancer cells. Restoring MIM expression completely blocked the pro-metastasis function of miR-182, while RhoA inhibition reversed the phenotypes of both miR-182 overexpression and MIM knockdown. In breast tumor samples, miR-182 induction is linked to downregulation of MIM, RhoA activation and poor prognosis. Hence, our data delineates the molecular pathway by which miR-182 promotes breast cancer invasion and metastasis, and may have important implication for the treatment of metastatic cancers.

Metadherin (MTDH), the newly discovered gene, is overexpressed in more than 40% of breast cancers. Recent studies have revealed that MTDH favors an oncogenic course and chemoresistance. With a number of breast cancer cell lines and breast tumor samples, we found that the relative expression of MTDH correlated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity in breast cancer. In this study, we found that knockdown of endogenous MTDH cells sensitized the MDA-MB-231 cells to TRAIL-induced apoptosis both in vitro and in vivo. Conversely, stable overexpression of MTDH in MCF-7 cells enhanced cell survival with TRAIL treatment. Mechanically, MTDH down-regulated caspase-8, decreased caspase-8 recruitment into the TRAIL death-inducing signaling complex, decreased caspase-3 and poly(ADP-ribose) polymerase-2 processing, increased Bcl-2 expression, and stimulated TRAIL-induced Akt phosphorylation, without altering death receptor status. In MDA-MB-231 breast cancer cells, sensitization to TRAIL upon MTDH down-regulation was inhibited by the caspase inhibitor Z-VAD-fmk (benzyloxycarbonyl-VAD-fluoromethyl ketone), suggesting that MTDH depletion stimulates activation of caspases. In MCF-7 breast cancer cells, resistance to TRAIL upon MTDH overexpression was abrogated by depletion of Bcl-2, suggesting that MTDH-induced Bcl-2 expression contributes to TRAIL resistance. We further confirmed that MTDH may control Bcl-2 expression partly by suppressing miR-16. Collectively, our results point to a protective function of MTDH against TRAIL-induced death, whereby it inhibits the intrinsic apoptosis pathway through miR-16-mediated Bcl-2 up-regulation and the extrinsic apoptosis pathway through caspase-8 down-regulation.

The application of functional genomic analysis of breast cancer metastasis has led to the identification of a growing number of organ-specific metastasis genes, which often function in concert to facilitate different steps of the metastatic cascade. However, the gene regulatory network that controls the expression of these metastasis genes remains largely unknown. Here, we demonstrate a computational approach for the deconvolution of transcriptional networks to discover master regulators of breast cancer bone metastasis. Several known regulators of breast cancer bone metastasis such as Smad4 and HIF1 were identified in our analysis. Experimental validation of the networks revealed BACH1, a basic leucine zipper transcription factor, as the common regulator of several functional metastasis genes, including MMP1 and CXCR4. Ectopic expression of BACH1 enhanced the malignance of breast cancer cells, and conversely, BACH1 knockdown significantly reduced bone metastasis. The expression of BACH1 and its target genes was linked to the higher risk of breast cancer recurrence in patients. This study established BACH1 as the master regulator of breast cancer bone metastasis and provided a paradigm to identify molecular determinants in complex pathological processes.

Bone metastasis is a frequent complication of breast cancer and a common cause of morbidity and mortality from the disease. During metastasis secreted proteins play crucial roles in the interactions between cancer cells and host stroma. To characterize the secreted proteins that are associated with breast cancer bone metastasis, we preformed a label-free proteomic analysis to compare the secretomes of four MDA-MB-231 (MDA231) derivative cell lines with varied capacities of bone metastasis. A total of 128 proteins were found to be consistently up-/down-regulated in the conditioned medium of bone-tropic cancer cells. The enriched molecular functions of the altered proteins included receptor binding and peptidase inhibition. Through additional transcriptomic analyses of breast cancer cells, we selected cystatin E/M (CST6), a cysteine protease inhibitor down-regulated in bone-metastatic cells, for further functional studies. Our results showed that CST6 suppressed the proliferation, colony formation, migration and invasion of breast cancer cells. The suppressive function against cancer cell motility was carried out by cancer cell-derived soluble CST6. More importantly, ectopic expression of CST6 in cancer cells rescued mice from overt osteolytic metastasis and deaths in the animal study, while CST6 knockdown markedly enhanced cancer cell bone metastasis and shortened animal survival. Overall, our study provided a systemic secretome analysis of breast cancer bone tropism and established secreted CST6 as a bona fide suppressor of breast cancer osteolytic metastasis.

ARF6 GTPase is an important regulator of membrane trafficking and actin-based cytoskeleton dynamics active at the leading edge of migrating cells. The integrin family heterodimeric transmembrane proteins serve as major receptors for extracellular matrix proteins, which play essential roles in cell adhesion and migration. Our recent proteomic analyses of ARF6 effectors have identified a novel ARF6 GTPase-activating protein, ACAP4, essential for EGF-induced cell migration. However, molecular mechanisms underlying ACAP4-mediated cell migration has remained elusive. Here we show that ACAP4 regulates integrin β1 dynamics during EGF-stimulated cell migration by interaction with Grb2. Our biochemical study shows that EGF stimulation induces phosphorylation of tyrosine 733 which enables ACAP4 to bind Grb2. This interaction of ACAP4 with Grb2 regulates integrin β1 recycling to the plasma membrane. Importantly, knockdown of ACAP4 by siRNA or overexpression of ACAP4 decreased recycling of integrin β1 to the plasma membrane and reduced integrin-mediated cell migration. Taken together, these results suggest a novel function for ACAP4 in the regulation of cell migration through controlling integrin β1 dynamics.