NSDHL promotes triple-negative breast cancer metastasis through the TGFβ signaling pathway and cholesterol biosynthesis

Breast Cancer Research and Treatment - Metastasis is the main cause of breast cancer mortality. Recent studies have proved that lipid metabolic reprogramming plays critical roles in breast cancer...     

LGR5 promotes the proliferation and tumor formation of cervical cancer cells through the Wnt/β-catenin signaling pathway

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a seven transmembrane receptor known as a potential stem cell marker for intestinal crypts and hair follicles, has recently been found to be overexpressed in some types of human cancers. However, the role of LGR5 in cervical cancer remains unclear. In this study, the expression of LGR5 gradually increases from normal cervix to cervical cancer in situ and to cervical cancers as revealed by immunohistochemistry and western blot analyses. Through knocking down or overexpressing LGR5 in SiHa and HeLa cells, the expression level of LGR5 was found to be positively related to cell proliferation in vitro and to tumor formation in vivo. Further investigation indicated that LGR5 protein could significantly promote the acceleration of cell cycle. Moreover, the TOP-Flash reporter assay and western blot for β-catenin, cyclinD1, and c-myc proteins, target genes of the Wnt/β-catenin pathway, indicated that LGR5 significantly activated Wnt/β-catenin signaling. Additionally, the blockage of Wnt/β-catenin pathway resulted in a significant inhibition of cell proliferation induced by LGR5. Taken together, these results demonstrate that LGR5 can promote proliferation and tumor formation in cervical cancer cells by activating the Wnt/β-catenin pathway.     

Alternariol induces DNA polymerase β expression through the PKA-CREB signaling pathway

Alternariol (AOH) is a mycotoxin of Alternaria alternata and can cause DNA damage and gene mutations. Low-dose and long-term treatment with AOH has been linked with incidence of esophageal carcinoma. DNA polymerase β (polβ) is a key enzyme in DNA base excision repair (BER). When it is overexpressed or mutated in cells, DNA polβ can cause genetic instability. Elevated DNA polβ has also been reported in several human cancers. Here, we report that AOH at 2, 10, 20 μM induces DNA polβ expression. In the process, protein kinase A (PKA) catalytic subunit activation, nuclear translocation and cAMP response element binding protein (CREB) phosphorylation are involved. AOH also increased CREB binding to the cAMP response element (CRE) consensus motif, which is present in the DNA polβ gene promoter. The PKA inhibitor H89 was able to block AOH-induced PKA-CREB activation, CREB DNA binding activity and decrease DNA polβ expression. Our results suggest that AOH can upregulate DNA polβ expression through the PKA-CREB signal transduction pathway.     

Slit2/Robo1 signaling promotes intestinal tumorigenesis through Src-mediated activation of the Wnt/β-catenin pathway

Slit2 is often overexpressed in cancers. Slit2 is a secreted protein that binds to Roundabout (Robo) receptors to regulate cell growth and migration. Here, we employed several complementary mouse models of intestinal cancers, including the Slit2 transgenic mice, the Apc^Min/+ spontaneous intestinal adenoma mouse model, and the DMH/DSS-induced colorectal carcinoma model to clarify function of Slit2/Robo1 signaling in intestinal tumorigenesis. We showed that Slit2 and Robo1 are overexpressed in intestinal tumors and may contribute to tumor generation. The Slit2/Robo1 signaling can induce precancerous lesions of the intestine and tumor progression. Ectopic expression of Slit2 activated Slit2/Robo1 signaling and promoted tumorigenesis and tumor growth. This was mediated in part through activation of the Src signaling, which then down-regulated E-cadherin, thereby activating Wnt/β-catenin signaling. Thus, Slit2/Robo1 signaling is oncogenic in intestinal tumorigenesis.     

CHC promotes tumor growth and angiogenesis through regulation of HIF-1α and VEGF signaling

Pancreatic adenocarcinoma is an aggressive disease with a high mortality rate. In this study, we have newly generated a monoclonal antibody (mAb), Pa65-2, which specifically binds to pancreatic cancer cells and tumor blood vessels. The target protein of Pa65-2 is identified as human clathrin heavy chain (CHC). In vitro and In vivo study showed that suppression of CHC either by shRNA or by Pa65-2 inhibited tumor growth and angiogenesis. One of the key functions of CHC was to bind with the hypoxia-inducing factor (HIF)-1α protein, increasing the stability of this protein and facilitating its nuclear translocation, thereby regulating the expression of VEGF. Taken together, our findings indicate that CHC plays a role in the processes of tumorigenesis and angiogenesis. Pa65-2 antibody or CHC shRNA can potentially be used for pancreatic cancer therapy.     

Lack of transforming growth factor-β signaling promotes collective cancer cell invasion through tumor-stromal crosstalk

ABSTRACT: INTRODUCTION: Transforming growth factor beta (TGF-β) has a dual role during tumor progression, initially as a suppressor and then as a promoter. Epithelial TGF-β signaling regulates fibroblast recruitment and activation. Concurrently, TGF-β signaling in stromal fibroblasts suppresses tumorigenesis in adjacent epithelia, while its ablation potentiates tumor formation. Much is known about the contribution of TGF-β signaling to tumorigenesis, yet the role of TGF-β in epithelial-stromal migration during tumor progression is poorly understood. We hypothesize that TGF-β is a critical regulator of tumor-stromal interactions that promote mammary tumor cell migration and invasion. METHODS: Fluorescently labeled murine mammary carcinoma cells, isolated from either MMTV-PyVmT transforming growth factor-beta receptor II knockout (TβRII KO) or TβRIIfl/fl control mice, were combined with mammary fibroblasts and xenografted onto the chicken embryo chorioallantoic membrane. These combinatorial xenografts were used as a model to study epithelial-stromal crosstalk. Intravital imaging of migration was monitored ex ovo, and metastasis was investigated in ovo. Epithelial RNA from in ovo tumors was isolated by laser capture microdissection and analyzed to identify gene expression changes in response to TGF-β signaling loss. RESULTS: Intravital microscopy of xenografts revealed that mammary fibroblasts promoted two migratory phenotypes dependent on epithelial TGF-β signaling: single cell/strand migration or collective migration. At epithelial-stromal boundaries, single cell/strand migration of TβRIIfl/fl carcinoma cells was characterized by expression of α-smooth muscle actin and vimentin, while collective migration of TβRII KO carcinoma cells was identified by E-cadherin+/p120+/β-catenin+ clusters. TβRII KO tumors also exhibited a twofold greater metastasis than TβRIIfl/fl tumors, attributed to enhanced extravasation ability. In TβRII KO tumor epithelium compared with TβRIIfl/fl epithelium, Igfbp4 and Tspan13 expression was upregulated while Col1α2, Bmp7, Gng11, Vcan, Tmeff1, and Dsc2 expression was downregulated. Immunoblotting and quantitative PCR analyses on cultured cells validated these targets and correlated Tmeff1 expression with disease progression of TGF-β-insensitive mammary cancer. CONCLUSION: Fibroblast-stimulated carcinoma cells utilize TGF-β signaling to drive single cell/strand migration but migrate collectively in the absence of TGF-β signaling. These migration patterns involve the signaling regulation of several epithelial-to-mesenchymal transition pathways. Our findings concerning TGF-β signaling in epithelial-stromal interactions are important in identifying migratory mechanisms that can be targeted as recourse for breast cancer treatment.     

Direct inhibition of the transforming growth factor-β pathway by protein-bound polysaccharide through inactivation of Smad2 signaling

Transforming growth factor-β (TGF-β) is involved in the regulation of cell proliferation, differentiation, and apoptosis and is associated with epithelial-mesenchymal transition (EMT). Inhibition of the TGF-β pathway is an attractive strategy for the treatment of cancer. We recently screened for novel TGF-β inhibitors among commercially available drugs and identified protein-bound polysaccharide (PSK) as a strong inhibitor of the TGF-β-induced reporter activity of 3TP-lux, a TGF-β1-responsive luciferase reporter. Protein-bound polysaccharide is used as a non-specific immunostimulant for the treatment of gastric and colorectal cancers in Japan. The anticancer activity of this agent may involve direct regulation of growth factor production and enzyme activity in tumors in addition to its immunomodulatory effect. Although several clinical studies have shown the beneficial therapeutic effects of PSK on various types of tumors, its mechanism of action is not clear. In the present study, Western blot analysis showed that PSK suppressed the phosphorylation and nuclear localization of the Smad2 protein, thereby suggesting that PSK inhibits the Smad and MAPK pathways. Quantitative PCR analysis showed that PSK decreased the expression of several TGF-β pathway target genes. E-cadherin and vimentin immunohistochemistry showed that PSK suppressed TGF-β1-induced EMT, and FACS analysis showed that PSK inhibited the EMT-mediated generation of CD44(+) /CD24(-) cells. These data provide new insights into the mechanisms mediating the TGF-β-inhibiting activity of PSK and suggest that PSK can effectively treat diseases associated with TGF-β signaling.     

Tumor-associated macrophage-derived transforming growth factor-β promotes colorectal cancer progression through HIF1-TRIB3 signaling

Tumor-associated macrophages (TAMs), one of the most common cell components in the tumor microenvironment, have been reported as key contributors to cancer-related inflammation and enhanced metastatic progression of tumors. To explore the underlying mechanism of TAM-induced tumor progression, TAMs were isolated from colorectal cancer patients, and the functional interaction with colorectal cancer cells was analyzed. Our study found that coculture of TAMs contributed to a glycolytic state in colorectal cancer, which promoted the stem-like phenotypes and invasion of tumor cells. TAMs produced the cytokine transforming growth factor-β to support hypoxia-inducible factor 1α (HIF1α) expression, thereby upregulating Tribbles pseudokinase 3 (TRIB3) in tumor cells. Elevated expression of TRIB3 resulted in activation of the β-catenin/Wnt signaling pathway, which eventually enhanced the stem-like phenotypes and cell invasion in colorectal cancer. Our findings provided evidence that TAMs promoted colorectal cancer progression in a HIF1α/TRIB3-dependent manner, and blockade of HIF1α signals efficiently improved the outcome of chemotherapy, describing an innovative approach for colorectal cancer treatment.     

CCN1 promotes tumorigenicity through Rac1/Akt/NF-κB signaling pathway in pancreatic cancer

Aberrant CCN1 expression has been reported to play an important role in the tumor development. However, the pattern and the role of CCN1 in pancreatic cancer remain largely unknown. Therefore, we further deciphered the role CCN1 played in pancreatic cancer. We first evaluated the CCN1 expression in human pancreatic cancer tissues and pancreatic cancer cells. Then we forced expression and silenced CCN1 expression in pancreatic cancer cell lines MIA PaCa2 and PANC-1 respectively, using lentivirus vectors. We characterized the stable cells in vitro and in vivo using a nude mouse xenograft model. In this study, we found that CCN1 expression was significantly higher in cancer specimens which positively correlated with the expression level of phosphorylated Akt and p65. and poorer outcome. Moreover, our results demonstrated that CCN1 positively regulated pancreatic cell growth in vitro and in vivo and helped cancer cells resist to tumor necrosis factor alpha-induced apoptosis. Furthermore, we disclosed that activation of CCN1/ras-related c3 botulinum toxin substrate 1 (Rac1)/V-akt murine thymoma viral oncogene homolog (Akt)/nuclear factor-kappa B pathway inhibited apoptosis in pancreatic cancer cells. CCN1 is upregulated in pancreatic cancer and promotes the survival of pancreatic cancer cells. Taken together, these results indicate that CCN1 may be a potential target for pancreatic cancer therapy.     

Estrogen upregulates the IGF-1 signaling pathway in lung cancer through estrogen receptor-β

The estrogen receptor (ER) signaling and the insulin-like growth factor-1 receptor (IGF-1R) signaling are implicated in lung cancer progression. Here, we sought to investigate whether estrogen regulated the IGF-1R signaling in non-small cell lung cancer (NSCLC) and the underlying mechanisms. We examined and analyzed the correlation of the expression of aromatase (Arom), ERβ, ERα, insulin-like growth factor-1 (IGF-1), and IGF-1R in NSCLC. Tissue-microarray and immunohistochemistry analysis of tissue specimens from 162 NSCLC patients and 38 patients with benign pulmonary lesions showed that Arom, ERβ, IGF-1, and IGF-1R were overexpressed while ERα was not expressed in NSCLC. Furthermore, ERβ expression was positively correlated with that of Arom, IGF-1, and IGF-1R (r = 0.554, 0.649, 0.496, respectively, P values are equal to 0.000), while Arom expression was positively associated with that of IGF-1 and IGF-1R (r = 0.657, 0.714, respectively, P values are equal to 0.000). Additionally, ERβ, IGF-1, and phospho-IGF-1R, but not ERα, were expressed in A549 cells. Immunoblotting assays showed that A549 cells treated with E2 showed significantly higher IGF-1 and p-IGF-1R levels than those receiving the combination treatment of 17β-estradiol (E2) and fulvestrant (Ful, ER antagonist) (P = 0.042, 0.002, respectively) or controls (P values are equal to 0.000). The MTT assays further revealed that E2 and IGF-1 synergistically promoted A549 cell proliferation. Together, our study provides the first direct evidence for an interaction between ER and IGF-1R in lung cancer. We showed that estrogen upregulated the IGF-1R signaling through ERβ in lung cancer tissues and A549 cells. These findings shed further light on the mechanisms whereby estrogen promotes lung cancer and highlight the ER and IGF-1R signaling pathways as promising targets for combinational therapy for lung cancer.     

MACC1 promotes carcinogenesis of colorectal cancer via β-catenin signaling pathway

Here we confirmed that metastasis-associated in colon cancer 1 (MACC1) and β-catenin expression were higher in colorectal cancer (CRC) cells and tissues than those in normal colonic epithelial cell line and adjacent non-tumour colorectal mucosa (ANM) tissues, respectively. MACC1 expression was significantly related to histological differentiation (p<0.001), UICC stage (p=0.029), T classification (p=0.017), and N classification (p=0.023). Cox regression analysis demonstrated that high MACC1/abnormal β-catenin expression was the strongest independent prognostic indicator for reduced overall survival in CRC patients. Significant positive correlation between MACC1 expression and abnormal β-catenin expression was found in CRC tissues. MACC1 knockdown dramatically inhibited cellular proliferation, migration, invasion, colony formation, and tumorigenesis, both in vitro and in vivo, but induced apoptosis in CRC cells. Further MACC1 over-expression increased Met, β-catenin, and its downstream genes including c-Myc, cyclin D1, and MMP9 expression, and its upstream gene phos-GSK3β (Ser9) expression. In addition, MACC1 increased vimentin and suppressed E-cadherin in HCT116 cells. Silencing of MACC1 reversed all these changes. Our results firstly suggest that MACC1 plays an important role in carcinogenesis and progression of CRC through β-catenin signaling pathway and mesenchymal-epithelial transition.     

Regulatory role of the transforming growth factor-β signaling pathway in the drug resistance of gastrointestinal cancers

Gastrointestinal (GI) cancer, including esophageal, gastric, and colorectal cancer, is one of the most prevalent types of malignant carcinoma and the leading cause of cancer-related deaths. Despite significant advances in therapeutic strategies for GI cancers in recent decades, drug resistance with various mechanisms remains the prevailing cause of therapy failure in GI cancers. Accumulating evidence has demonstrated that the transforming growth factor (TGF)-β signaling pathway has crucial, complex roles in many cellular functions related to drug resistance. This review summarizes current knowledge regarding the role of the TGF-β signaling pathway in the resistance of GI cancers to conventional chemotherapy, targeted therapy, immunotherapy, and traditional medicine. Various processes, including epithelial-mesenchymal transition, cancer stem cell development, tumor microenvironment alteration, and microRNA biogenesis, are proposed as the main mechanisms of TGF-β-mediated drug resistance in GI cancers. Several studies have already indicated the benefit of combining antitumor drugs with agents that suppress the TGF-β signaling pathway, but this approach needs to be verified in additional clinical studies. Moreover, the identification of potential biological markers that can be used to predict the response to TGF-β signaling pathway inhibitors during anticancer treatments will have important clinical implications in the future.     

BMK1 kinase suppresses epithelial-mesenchymal transition through the Akt/GSK3β signaling pathway

Epithelial-mesenchymal transition (EMT) plays a crucial role in the development of cancer metastasis. The mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase, c-jun-NH(2)-kinase, and p38 have been implicated in promoting EMT, but a role for the MAP kinase BMK1 has not been studied. Here, we report that BMK1 signaling suppresses EMT. BMK1 elevation augmented E-cadherin-mediated cell-cell adhesion, downregulated mesenchymal markers, and decreased cell motility. Conversely, BMK1 silencing attenuated E-cadherin-mediated cell-cell adhesion, upregulated mesenchymal markers, and stimulated cell motility. BMK1 depletion dramatically increased the accumulation of endogenous Snail in the nuclear compartment. Snail accumulation was mediated by Akt/GSK3β signaling, which was activated by a modulation in the expression of the mTOR inhibitor DEPTOR. In support of these observations, BMK1 depletion promoted metastasis in vivo. Together, our findings reveal a novel mechanism of EMT control via mTOR/Akt inhibition that suppresses cancer metastasis.     

Acidosis promotes invasiveness of breast cancer cells through ROS-AKT-NF-κB pathway

It is well known that acidic microenvironment promotes tumorigenesis, however, the underlying mechanism remains largely unknown. In the present study, we show that acidosis promotes invasiveness of breast cancer cells through a series of signaling events. First, our study indicates that NF-κB is a key factor for acidosis-induced cell invasion. Acidosis activates NF-κB without affecting STAT3 activity; knockdown of NF-κB p65 abrogates the acidosis-induced invasion activity. Next, we show that the activation of NF-κB is mediated through phosphorylation and degradation of IκBα; and phosphorylation and nuclear translocation of p65. Upstream to NF-κB signaling, AKT is activated under acidic conditions. Moreover, acidosis induces generation of reactive oxygen species (ROS) which can be suppressed by ROS scavengers, reversing the acidosis-induced activation of AKT and NF-κB, and invasiveness. As a negative regulator of AKT, PTEN is oxidized and inactivated by the acidosis-induced ROS. Finally, inhibition of NADPH oxidase (NOX) suppresses acidosis-induced ROS production, suggesting involvement of NOX in acidosis-induced signaling cascade. Of considerable interest, acidosis-induced ROS production and activation of AKT and NF-κB can be only detected in cancer cells, but not in non-malignant cells. Together, these results demonstrate a cancer specific acidosis-induced signaling cascade in breast cancer cells, leading to cell invasion.     

Estrogen and antiestrogens alter breast cancer invasiveness by modulating the transforming growth factor-β signaling pathway

In the later stages of breast cancer, estrogen receptor (ER)α-negative cancers typically have higher histological grades than ERα-positive cancers, and transforming growth factor (TGF)-β promotes invasion and metastasis. Our previous study indicated that ERα inhibited TGF-β signaling by inducing the degradation of Smad in an estrogen-dependent manner. In the present study, we report that the suppressive effects of ERα and estrogen on tumor progression are mediated by inhibiting TGF-β signaling. Furthermore, we investigated the effects of antiestrogens such as ICI182,780 (ICI) or tamoxifen (TAM) on TGF-β signaling and breast cancer invasiveness. The levels of total Smad and pSmad were reduced by estrogen, whereas ICI slightly increased them, and TAM had no effect. To investigate the effect of antiestrogens on breast cancer invasiveness, we generated highly migratory and invasive MCF-7-M5 cells. The migration and invasion of these cells were suppressed by the inhibitor of TGF-β receptor kinase, SB-505124, and estrogen. However, antiestrogens did not suppress the migration and invasion of these cells. In addition, we screened TGF-β target genes whose expression was reduced by estrogen treatment and identified four genes associated with breast cancer invasiveness and poor prognosis. The expression of these genes was not decreased by antiestrogens. These observations provide a new insight into estrogen function and the mechanisms underlying estrogen-mediated suppression of tumor progression.     

PTEN activation sensitizes breast cancer to PI3-kinase inhibitor through the β-catenin signaling pathway

Combination therapy is considered a promising therapeutic modality in enhancing treatment efficacy. The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is almost universally dysregulated in breast cancer, with specific occurrence of PTEN mutations; thus, it has become an attractive target for cancer treatment. However, the use of single targeted therapeutics against the PI3K/AKT pathway has demonstrated only modest clinical benefits. In this study, recombinant adenovirus-mediated gene transfer of PTEN (AD-PTEN) combined with treatment with LY294002 was utilized to evaluate the effects of suppression of breast cancer cell proliferation. Herein, we show that AD-PTEN significantly enhanced the sensitization of breast cancer cells to LY294002. The 50% inhibitory concentration (IC50) values of LY294002 were significantly decreased to a greater extent in cells transfected with combination therapy. In addition, treatment of AD-PTEN-transfected cells with LY294002 resulted in significantly reduced cell viability and invasion ability compared to single LY294002 treatment. Using western blotting, we found that combination treatment resulted in lower levels of phosphorylated AKTSer473 and GSK-3βSer9 than single treatment with LY294002. Furthermore, we showed a significant decrease in nuclear β-catenin, Fra-1, Tcf-4 and c-Myc by combination treatment. Our results indicate that AD-PTEN sensitization of breast cancer to LY294002 is achieved by increased GSK-3β activity, thus resulting in inhibition of the β-catenin signaling pathway.