cAMP-response-element-binding protein positively regulates breast cancer metastasis and subsequent bone destruction

cAMP-response-element-binding protein (CREB) signaling has been reported to be associated with cancer development and poor clinical outcome in various types of cancer. However, it remains to be elucidated whether CREB is involved in breast cancer development and osteotropism. Here, we found that metastatic MDA-MB-231 breast cancer cells exhibited higher CREB expression than did non-metastatic MCF-7 cells and that CREB expression was further increased by several soluble factors linked to cancer progression, such as IL-1, IGF-1, and TGF-β. Using wild-type CREB and a dominant-negative form (K-CREB), we found that CREB signaling positively regulated the proliferation, migration, and invasion of MDA-MB-231 cells. In addition, K-CREB prevented MDA-MB-231 cell-induced osteolytic lesions in a mouse model of cancer metastasis. Furthermore, CREB signaling in cancer cells regulated the gene expression of PTHrP, MMPs, and OPG, which are closely involved in cancer metastasis and bone destruction. These results indicate that breast cancer cells acquire CREB overexpression during their development and that this CREB upregulation plays an important role in multiple steps of breast cancer bone metastasis.     

Upregulated microRNA-301a in breast cancer promotes tumor metastasis by targeting PTEN and activating Wnt/β-catenin signaling

MicroRNAs (miRNAs) are strongly implicated in many cancers, including breast cancer. Recently, microRNA-301a (miR-301a) has been proved to play a substantial role in gastric cancer, but its functions in the context of breast cancer remain unknown. Here we report that miR-301a was markedly upregulated in primary tumor samples from patients with distant metastases and pro-metastatic breast cancer cell lines. Gain-of-function and loss-of-function studies showed that ectopic overexpression of miR-301a promoted breast cancer cell migration, invasion and metastasis both in vitro and in vivo. Notably, Wnt/β-catenin signaling was hyperactivated in metastatic breast cancer cells that express miR-301a, and mediated miR-301a-induced invasion and metastasis. Furthermore, miR-301a directly targeted and suppressed PTEN, one negative regulator of the Wnt/β-catenin signaling cascade. These results demonstrate that miR-301a maintains constitutively activated Wnt/β-catenin signaling by directly targeting PTEN, which promotes breast cancer invasion and metastasis. Taken together, our findings reveal a new regulatory mechanism of miR-301a and suggest that miR-301a might be a potential target in breast cancer therapy.     

Adipocyte-derived IL-6 and leptin promote breast Cancer metastasis via upregulation of Lysyl Hydroxylase-2 expression

Background

Adipocytes make up the major component of breast tissue, accounting for 90% of stromal tissue. Thus, the crosstalk between adipocytes and breast cancer cells may play a critical role in cancer progression. Adipocyte-breast cancer interactions have been considered important for the promotion of breast cancer metastasis. However, the specific mechanisms underlying these interactions are unclear. In this study, we investigated the mechanisms of adipocyte-mediated breast cancer metastasis.

Methods

Breast cancer cells were cocultured with mature adipocytes for migration and 3D matrix invasion assays. Next, lentivirus-mediated loss-of-function experiments were used to explore the function of lysyl hydroxylase (PLOD2) in breast cancer migration and adipocyte-dependent migration of breast cancer cells. The role of PLOD2 in breast cancer metastasis was further confirmed using orthotopic mammary fat pad xenografts in vivo. Clinical samples were used to confirm that PLOD2 expression is increased in tumor tissue and is associated with poor prognosis of breast cancer patients. Cells were treated with cytokines and pharmacological inhibitors in order to verify which adipokines were responsible for activation of PLOD2 expression and which signaling pathways were activated in vitro.

Results

Gene expression profiling and Western blotting analyses revealed that PLOD2 was upregulated in breast cancer cells following coculture with adipocytes; this process was accompanied by enhanced breast cancer cell migration and invasion. Loss-of-function studies indicated that PLOD2 knockdown suppressed cell migration and disrupted the formation of actin stress fibers in breast cancer cells and abrogated the migration induced by following coculture with adipocytes. Moreover, experiments performed in orthotopic mammary fat pad xenografts showed that PLOD2 knockdown could reduce metastasis to the lung and liver. Further, high PLOD2 expression correlated with poor prognosis of breast cancer patients. Mechanistically, adipocyte-derived interleukin-6 (IL-6) and leptin may facilitate PLOD2 upregulation in breast cancer cells and promote breast cancer metastasis in tail vein metastasis assays. Further investigation revealed that adipocyte-derived IL-6 and leptin promoted PLOD2 expression through activation of the JAK/STAT3 and PI3K/AKT signaling pathways.

Conclusions

Our study reveals that adipocyte-derived IL-6 and leptin promote PLOD2 expression by activating the JAK/STAT3 and PI3K/AKT signaling pathways, thus promoting breast cancer metastasis.

     

Role of Bmi-1 in Regulation of Ionizing Irradiation-Induced Epithelial-Mesenchymal Transition and Migration of Breast Cancer Cells

Radiotherapy is a widely used treatment for cancer. However, recent studies suggest that ionizing radiation (IR) can promote tumor invasion and metastasis. Bmi-1, a member of the polycomb group protein family, has been observed as a regulator of oxidative stress and promotes metastasis in some tumors. But, its potential role in the metastasis induced by IR of breast cancer has not been explored. In our study, we found that increased levels of Bmi-1 were correlated to EMT of breast cancer cells. Through analyzing the EMT state and metastasis of breast cancer induced by IR, we found the metastatic potential of breast cancer cells can either be inhibited or accelerated by IR following a time-dependent pattern. Silencing Bmi-1 completely abolished the ability of the IR to alter, reduce or increase, the migration of breast cancer cells. Also, when Bmi-1 was knocked down, the effect of inhibition of PI3K/AKT signaling on EMT affected by IR was blocked. These results suggest that Bmi-1 is a key gene in regulation of EMT and migration of breast cancer cells induced by IR through activation of PI3K/AKT signaling; therefore, Bmi-1 could be a new target for inhibiting metastasis caused by IR.     

Identification of novel metastasis suppressor signaling pathways for breast cancer

Cancer lethality is mainly caused by metastasis. Therefore, understanding the nature of the genes involved in this process has become a priority. Given the heterogeneity of mutations in cancer cells, considerable focus has been directed toward characterizing metastasis genes in the context of relevant signaling pathways rather than treating genes as independent and equal entities. One signaling cascade implicated in the regulation of cell growth, invasion and metastasis is the MAP kinase pathway. Raf kinase inhibitory protein (RKIP) functions as an inhibitor of the MAP kinase pathway and is a metastasis suppressor in different cancer models. By utilizing statistical analysis of clinical data integrated with experimental validation, we recently identified components of the RKIP signaling pathway relevant to breast cancer metastasis. Using the RKIP pathway as an example, we show how prior biological knowledge can be efficiently combined with genome-wide patient data to identify gene regulatory mechanisms that control metastasis.     

Denbinobin suppresses breast cancer metastasis through the inhibition of Src-mediated signaling pathways

Denbinobin (5-hydroxy-3,7-dimethoxy- 1,4-phenanthraquinone), a biologically active chemical isolated from Ephemerantha lonchophylla, has been demonstrated to display anti-cancer activity. Breast cancer is the leading cause of female mortality, and the high mortality is mainly attributable to metastasis. Src kinase activity is elevated in many human cancers, including breast cancer, and is often associated with aggressive disease. In the present study, we examined the anti-metastatic effects of denbinobin through decreasing Src kinase activity in human and mouse breast cancer cells. Denbinobin caused significant block of Src kinase activity in both human and mouse breast cancer cells. Moreover, phosphorylation of the signaling molecules focal adhesion kinase, Crk-associated substrate and paxillin downstream of Src was also inhibited by denbinobin. Furthermore, denbinobin inhibited the in vitro migration, invasion and in vivo metastasis of breast cancers in a mouse metastatic model. The denbinobin-treated group showed a significant reduction in tumor metastasis, orthrotopic tumor volume, and spleen enlargement compared to the control group. In addition, transfection of breast cancer cells with a plasmid coding for a constitutively active Src prevented the denbinobin-mediated phosphorylation of Src and downstream molecules and cell migration. Our findings provide evidences that denbinobin inhibits Src-mediated signaling pathways involved in controlling breast cancer migration and metastasis, suggesting that it has therapeutic potential in breast cancer treatment.     

Identification of novel metastasis suppressor signaling pathways for breast cancer

Cancer lethality is mainly caused by metastasis. Therefore, understanding the nature of the genes involved in this process has become a priority. Given the heterogeneity of mutations in cancer cells, considerable focus has been directed toward characterizing metastasis genes in the context of relevant signaling pathways rather than treating genes as independent and equal entities. One signaling cascade implicated in the regulation of cell growth, invasion and metastasis is the MAP kinase pathway. Raf kinase inhibitory protein (RKIP) functions as an inhibitor of the MAP kinase pathway and is a metastasis suppressor in different cancer models. By utilizing statistical analysis of clinical data integrated with experimental validation, we recently identified components of the RKIP signaling pathway relevant to breast cancer metastasis. Using the RKIP pathway as an example, we show how prior biological knowledge can be efficiently combined with genome-wide patient data to identify gene regulatory mechanisms that control metastasis.     

Delta/notch-like epidermal growth factor-related receptor promotes stemness to facilitate breast cancer progression

DNER, Delta/Notch-like epidermal growth factor (EGF)-related receptor, is a neuron-specific transmembrane protein carrying extracellular EGF-like repeats. The function of DNER in breast cancer has not been evaluated. The present study demonstrates that the expression of DNER in breast cancer tissue is significantly higher than its expression in breast benign disease and is associated with poor recurrence-free survival (RFS) of breast cancer patients. It demonstrated that DNER could enhance the proliferation and metastasis of breast cancer cells in vitro and significantly increases tumor growth in vivo. Our study uncovered that DNER can promote breast cancer cells proliferation and metastasis by activating Girdin/PI3K/AKT signaling and subsequently regulating several key genes involving the characters of cancer stem cells. Taken together, DNER promotes breast cancer growth and metastasis, which provided a theoretical basis for future applications of DNER inhibitors in the treatment of breast cancer.     

Stabilization of Notch1 and β-catenin in response to ER- breast cancer-specific up-regulation of PSAT1 mediates distant metastasis

Breast cancer has the highest incidence in women worldwide, with a mortality rate second only to lung cancer. Distant metastasis is the major cause of breast cancer-induced death. While upregulation of phosphoserine aminotransferase 1 (PSAT1) has been reported in several cancer types, its specific roles in breast cancer and potential involvement in distant metastasis remain unclear. In our study, PSAT1 was upregulated in metastatic breast cancer and promoted distant metastasis both in vitro and in vivo. Data obtained from transwell and wound healing, colony, sphere assays and detection of various malignant phenotypic markers showed that PSAT1 mediates distant metastasis by promoting invasion, migration, proliferation, anti-apoptosis, stemness and angiogenesis in breast cancer cells. Mechanistically, PSAT1 activated Notch and β-catenin signaling pathways, leading to enhanced distant metastasis. The clinical relevance of PSAT1 in breast cancer was additionally investigated, which revealed associations of poorer patient prognosis with high PSAT1 mRNA and protein expression. In summary, PSAT1 is a critical molecular regulator of distant metastasis that may effectively serve as a marker of poor prognosis in breast cancer.     

Hedgehog信号通路与乳腺癌

近年来的研究表明,Hedgehog信号通路在肿瘤的发生发展中具有重要的作用,该通路基因突变或异常表达将导致多种器官肿瘤的发生,并与Wnt、MAPK等信号通路相互作用,共同调节肿瘤的发生发展。我们简要综述了Hedgehog信号通路在乳腺癌发生发展中的重要作用,旨在了解乳腺癌发生、发展的分子机制.     

Nuclear import of PTPN18 inhibits breast cancer metastasis mediated by MVP and importin β2

Distant metastasis is the primary cause of breast cancer-associated death. The existing information, such as the precise molecular mechanisms and effective therapeutic strategies targeting metastasis, is insufficient to combat breast cancer. This study demonstrates that the protein tyrosine phosphatase PTPN18 is downregulated in metastatic breast cancer tissues and is associated with better metastasis-free survival. Ectopic expression of PTPN18 inhibits breast cancer cell metastasis. PTPN18 is translocated from the cytoplasm to the nucleus by MVP and importin β2 in breast cancer. Then, nuclear PTPN18 dephosphorylates ETS1 and promotes its degradation. Moreover, nuclear PTPN18 but not cytoplasmic PTPN18 suppresses transforming growth factor-β signaling and epithelial-to-mesenchymal transition by targeting ETS1. Our data highlight PTPN18 as a suppressor of breast cancer metastasis and provide an effective antimetastatic therapeutic strategy.Subject terms: Breast cancer, Epithelial-mesenchymal transition     

Deglycosylation of epithelial cell adhesion molecule affects epithelial to mesenchymal transition in breast cancer cells

The transmembrane glycoprotein epithelial cell adhesion molecule (EpCAM) is overexpressed in most epithelial cancers including breast cancer, where it plays an important role in cancer progression. Previous study has demonstrated that knockdown of EpCAM inhibits breast cancer cell growth and metastasis via inhibition of the Ras/Raf/ERK signaling pathway and matrix metallopeptidase-9 (MMP-9). Although glycosylation is believed to be associated with the function of EpCAM, the contribution of N-glycosylation to this function remains unclear. We constructed the N-glycosylation mutation plasmid of EpCAM and used it to treat breast cancer cells. Loss of N-glycosylation at all three sites EpCAM had no effect on its level of expression or membrane localization. However, mutation at glycosylation sites significantly reduced the ability of EpCAM to promote epithelial to mesenchymal transition in breast cancer. N-glycosylation mutation of EpCAM led to decrease phosphorylation of Raf, ERK, and Akt, and inhibited the Ras/Raf/ERK and PI3K/Akt signaling pathways. Furthermore, we demonstrated that N-glycosylation mutation of EpCAM-mediated invasion and metastasis of breast carcinoma cells required the downregulation of MMP-9 via inhibition of these two signaling pathways. Our results identified the characteristics and function of EpCAM glycosylation. These data could illuminate molecular regulation of EpCAM by glycosylation and promote our understanding of the application of glycosylated EpCAM as a target for breast cancer therapy.     

Circular RNA circBCBM1 promotes breast cancer brain metastasis by modulating miR-125a/BRD4 axis

Circular RNAs (circRNAs) play critical roles in tumorigenesis and the progression of various cancers. We previously identified a novel upregulated circRNA, circBCBM1 (hsa_circ_0001944), in the context of breast cancer brain metastasis. However, the potential biological function and molecular mechanism of circBCBM1 in breast cancer brain metastasis remain largely unknown. In this study, we confirmed that circBCBM1 was a stable and cytoplasmic circRNA. Functionally, circBCBM1 promoted the proliferation and migration of 231-BR cells in vitro and growth and brain metastasis in vivo. Mechanistically, circBCBM1 acted as an endogenous miR-125a sponge to inhibit miR-125a activity, resulting in the upregulation of BRD4 (bromodomain containing 4) and subsequent upregulation of MMP9 (matrix metallopeptidase 9) through Sonic hedgehog (SHH) signaling pathway. Importantly, circBCBM1 was markedly upregulated in the breast cancer brain metastasis cells and clinical tissue and plasma samples; besides, circBCBM1 overexpression in primary cancerous tissues was associated with shorter brain metastasis-free survival (BMFS) of breast cancer patients. These findings indicate that circBCBM1 is involved in breast cancer brain metastasis via circBCBM1/miR-125a/BRD4 axis. CircBCBM1 may serve as a novel diagnostic and prognostic biomarker and potential therapeutic target for breast cancer brain metastasis.     

Stimulation of host bone marrow stromal cells by sympathetic nerves promotes breast cancer bone metastasis in mice

Bone and lung metastases are responsible for the majority of deaths in patients with breast cancer. Following treatment of the primary cancer, emotional and psychosocial factors within this population precipitate time to recurrence and death, however the underlying mechanism(s) remain unclear. Using a mouse model of bone metastasis, we provide experimental evidence that activation of the sympathetic nervous system, which is one of many pathophysiological consequences of severe stress and depression, promotes MDA-231 breast cancer cell colonization of bone via a neurohormonal effect on the host bone marrow stroma. We demonstrate that induction of RANKL expression in bone marrow osteoblasts, following β2AR stimulation, increases the migration of metastatic MDA-231 cells in vitro, independently of SDF1-CXCR4 signaling. We also show that the stimulatory effect of endogenous (chronic stress) or pharmacologic sympathetic activation on breast cancer bone metastasis in vivo can be blocked with the β-blocker propranolol, and by knockdown of RANK expression in MDA-231 cells. These findings indicate that RANKL promotes breast cancer cell metastasis to bone via its pro-migratory effect on breast cancer cells, independently of its effect on bone turnover. The emerging clinical implication, supported by recent epidemiological studies, is that βAR-blockers and drugs interfering with RANKL signaling, such as Denosumab, could increase patient survival if used as adjuvant therapy to inhibit both the early colonization of bone by metastatic breast cancer cells and the initiation of the "vicious cycle" of bone destruction induced by these cells.     

Exploring metabolic pathway disruption in the subchronic phencyclidine model of schizophrenia with the Generalized Singular Value Decomposition

Background

One of the major goals in gene and protein expression profiling of cancer is to identify biomarkers and build classification models for prediction of disease prognosis or treatment response. Many traditional statistical methods, based on microarray gene expression data alone and individual genes' discriminatory power, often fail to identify biologically meaningful biomarkers thus resulting in poor prediction performance across data sets. Nonetheless, the variables in multivariable classifiers should synergistically interact to produce more effective classifiers than individual biomarkers.

Results

We developed an integrated approach, namely network-constrained support vector machine (netSVM), for cancer biomarker identification with an improved prediction performance. The netSVM approach is specifically designed for network biomarker identification by integrating gene expression data and protein-protein interaction data. We first evaluated the effectiveness of netSVM using simulation studies, demonstrating its improved performance over state-of-the-art network-based methods and gene-based methods for network biomarker identification. We then applied the netSVM approach to two breast cancer data sets to identify prognostic signatures for prediction of breast cancer metastasis. The experimental results show that: (1) network biomarkers identified by netSVM are highly enriched in biological pathways associated with cancer progression; (2) prediction performance is much improved when tested across different data sets. Specifically, many genes related to apoptosis, cell cycle, and cell proliferation, which are hallmark signatures of breast cancer metastasis, were identified by the netSVM approach. More importantly, several novel hub genes, biologically important with many interactions in PPI network but often showing little change in expression as compared with their downstream genes, were also identified as network biomarkers; the genes were enriched in signaling pathways such as TGF-beta signaling pathway, MAPK signaling pathway, and JAK-STAT signaling pathway. These signaling pathways may provide new insight to the underlying mechanism of breast cancer metastasis.

Conclusions

We have developed a network-based approach for cancer biomarker identification, netSVM, resulting in an improved prediction performance with network biomarkers. We have applied the netSVM approach to breast cancer gene expression data to predict metastasis in patients. Network biomarkers identified by netSVM reveal potential signaling pathways associated with breast cancer metastasis, and help improve the prediction performance across independent data sets.     

Divergent Roles for IRS-1 and IRS-2 in Breast Cancer Metastasis

The insulin receptor substrate (IRS) proteins are cytoplasmic docking proteins that function as essential signaling intermediates downstream of activated cell surface receptors, many of which have been implicated in breast cancer. The IRS proteins do not contain intrinsic kinase activity but rather function by organizing signaling complexes to initiate intracellular signaling cascades. IRS-1 and IRS-2 are expressed in normal mammary epithelial cells and in breast carcinoma cells, where they have been implicated in mediating signals to promote tumor cell survival, growth and motility. Although IRS-1 and IRS-2 are homologous, recent studies have revealed distinct functions for these adaptor proteins in regulating breast cancer progression. Specifically, IRS-2 is a positive regulator of metastasis, whereas IRS-1 may be a suppressor of metastasis. The observation that IRS-1 is inactivated in metastatic mammary tumors raises the possibility that IRS activity, rather than expression, may be a novel predictive indicator of metastasis. Understanding how the IRS proteins function in tumor progression is essential for future efforts aimed at developing approaches to target IRS-1 and IRS-2 in a diagnostic or therapeutic manner for the benefit of breast cancer patients.     

A critical role of Gbetagamma in tumorigenesis and metastasis of breast cancer

A growing body of evidence indicates that G protein-coupled receptors (GPCRs) are involved in breast tumor progression and that targeting GPCRs may be a novel adjuvant strategy in cancer treatment. However, due to the redundant role of multiple GPCRs in tumor development, it may be necessary to target a common signaling component downstream of these receptors to achieve maximum efficacy. GPCRs transmit signals through heterotrimeric G proteins composed of Gα and Gβγ subunits. Here we evaluated the role of Gβγ in breast tumor growth and metastasis both in vitro and in vivo. Our data show that blocking Gβγ signaling with Gα(t) or small molecule inhibitors blocked serum-induced breast tumor cell proliferation as well as tumor cell migration induced by various GPCRs in vitro. Moreover, induced expression of Gα(t) in MDA-MB-231 cells inhibited primary tumor formation and retarded growth of existing breast tumors in nude mice. Blocking Gβγ signaling also dramatically reduced the incidence of spontaneous lung metastasis from primary tumors and decreased tumor formation in the experimental lung metastasis model. Additional studies indicate that Gβγ signaling may also play a role in the generation of a tumor microenvironment permissive for tumor progression, because the inhibition of Gβγ signaling attenuated leukocyte infiltration and angiogenesis in primary breast tumors. Taken together, our data demonstrate a critical role of Gβγ signaling in promoting breast tumor growth and metastasis and suggest that targeting Gβγ may represent a novel therapeutic approach for breast cancer.     

Kindlin-2 promotes Src-mediated tyrosine phosphorylation of androgen receptor and contributes to breast cancer progression

Androgen receptor (AR) signaling plays important roles in breast cancer progression. We show here that Kindlin-2, a focal adhesion protein, is critically involved in the promotion of AR signaling and breast cancer progression. Kindlin-2 physically associates with AR and Src through its two neighboring domains, namely F1 and F0 domains, resulting in formation of a Kindlin-2-AR-Src supramolecular complex and consequently facilitating Src-mediated AR Tyr-534 phosphorylation and signaling. Depletion of Kindlin-2 was sufficient to suppress Src-mediated AR Tyr-534 phosphorylation and signaling, resulting in diminished breast cancer cell proliferation and migration. Re-expression of wild-type Kindlin-2, but not AR-binding-defective or Src-binding-defective mutant forms of Kindlin-2, in Kindlin-2-deficient cells restored AR Tyr-534 phosphorylation, signaling, breast cancer cell proliferation and migration. Furthermore, re-introduction of phosphor-mimic mutant AR-Y534D, but not wild-type AR reversed Kindlin-2 deficiency-induced inhibition of AR signaling and breast cancer progression. Finally, using a genetic knockout strategy, we show that ablation of Kindlin-2 from mammary tumors in mouse significantly reduced AR Tyr-534 phosphorylation, breast tumor progression and metastasis in vivo. Our results suggest a critical role of Kindlin-2 in promoting breast cancer progression and shed light on the molecular mechanism through which it functions in this process.Subject terms: Cell signalling, Breast cancer