Jagged-1 and Notch3 juxtacrine loop regulates ovarian tumor growth and adhesion

Notch3 gene amplification and pathway activation have been reported in ovarian serous carcinoma. However, the primary Notch3 ligand that initiates signal transduction in ovarian cancer remains unclear. In this report, we identify Jagged-1 as the highest expressed Notch ligand in ovarian tumor cells as well as in peritoneal mesothelial cells that are in direct contact with disseminated ovarian cancer cells. Cell-cell adhesion and cellular proliferation were reduced in Notch3-expressing ovarian cancer cells that were cocultured with Jagged-1 knockdown mesothelial and tumor feeder cells. Interaction of Notch3-expressing ovarian cancer cells with Jagged-1-expressing feeder cells activated the promoter activity of candidate Notch3 target genes, and this activity was attenuated by Notch3 siRNA. Constitutive expression of the Notch3 intracellular domain significantly suppressed the Jagged-1 shRNA-mediated growth inhibitory effect. In Notch3-expressing ovarian cancer cells, Jagged-1-stimulating peptides enhanced cellular proliferation, which was suppressed by gamma-secretase inhibitor and Notch3 siRNA. Taken together, our results show that Jagged-1 is the primary Notch3 ligand in ovarian carcinoma and Jagged-1/Notch3 interaction constitutes a juxtacrine loop promoting proliferation and dissemination of ovarian cancer cells within the intraperitoneal cavity.     

The von Hippel-Lindau tumor suppressor protein regulates gene expression and tumor growth through histone demethylase JARID1C

In clear-cell renal cell carcinoma (ccRCC), inactivation of the tumor suppressor von Hippel-Lindau (VHL) occurs in the majority of the tumors and is causal for the pathogenesis of ccRCC. Recently, a large-scale genomic sequencing study of ccRCC tumors revealed that enzymes that regulate histone H3 lysine 4 trimethylation (H3K4Me3), such as JARID1C/KDM5C/SMCX and MLL2, were mutated in ccRCC tumors, suggesting that H3K4Me3 might have an important role in regulating gene expression and tumorigenesis. In this study we report that in VHL-deficient ccRCC cells, the overall H3K4Me3 levels were significantly lower than that of VHL+/+ counterparts. Furthermore, this was hypoxia-inducible factor (HIF) dependent, as depletion of HIF subunits by small hairpin RNA in VHL-deficient ccRCC cells restored H3K4Me3 levels. In addition, we demonstrated that only loss of JARID1C, not JARID1A or JARID1B, abolished the difference of H3K4Me3 levels between VHL-/- and VHL+/+ cells, and JARID1C displayed HIF-dependent expression pattern. JARID1C in VHL-/- cells was responsible for the suppression of HIF-responsive genes insulin-like growth factor-binding protein 3 (IGFBP3), DNAJC12, COL6A1, growth and differentiation factor 15 (GDF15) and density-enhanced phosphatase 1. Consistent with these findings, the H3K4Me3 levels at the promoters of IGFBP3, DNAJC12, COL6A1 and GDF15 were lower in VHL-/- cells than in VHL+/+ cells, and the differences disappeared after JARID1C depletion. Although HIF2α is an oncogene in ccRCC, some of its targets might have tumor suppressive activity. Consistent with this, knockdown of JARID1C in 786-O VHL-/- ccRCC cells significantly enhanced tumor growth in a xenograft model, suggesting that JARID1C is tumor suppressive and its mutations are tumor promoting in ccRCC. Thus, VHL inactivation decreases H3K4Me3 levels through JARID1C, which alters gene expression and suppresses tumor growth.     

Insulin-like growth factor-binding protein-2 promotes prostate cancer cell growth via IGF-dependent or -independent mechanisms and reduces the efficacy of docetaxel

Background:

The development of androgen independence, chemo-, and radioresistance are critical markers of prostate cancer progression and the predominant reasons for its high mortality. Understanding the resistance to therapy could aid the development of more effective treatments.

Aim:

The aim of this study is to investigate the effects of insulin-like growth factor-binding protein-2 (IGFBP-2) on prostate cancer cell proliferation and its effects on the response to docetaxel.

Methods:

DU145 and PC3 cells were treated with IGFBP-2, insulin-like growth factor I (IGF-I) alone or in combination with blockade of the IGF-I receptor or integrin receptors. Cells were also treated with IGFBP-2 short interfering ribonucleic acid with or without a PTEN (phosphatase and tensin homologue deleted on chromosome 10) inhibitor or docetaxel. Tritiated thymidine incorporation was used to measure cell proliferation and Trypan blue cell counting for cell death. Levels of IGFBP-2 mRNA were measured using RT–PCR. Abundance and phosphorylation of proteins were assessed using western immunoblotting.

Results:

The IGFBP-2 promoted cell growth in both cell lines but with PC3 cells this was in an IGF-dependent manner, whereas with DU145 cells the effect was independent of IGF receptor activation. This IGF-independent effect of IGFBP-2 was mediated by interaction with β-1-containing integrins and a consequent increase in PTEN phosphorylation. We also determined that silencing IGFBP-2 in both cell lines increased the sensitivity of the cells to docetaxel.

Conclusion:

The IGFBP-2 has a key role in the growth of prostate cancer cells, and silencing IGFBP-2 expression reduced the resistance of these cells to docetaxel. Targeting IGFBP-2 may increase the efficacy of docetaxel.     

Notch signaling regulates tumor angiogenesis by diverse mechanisms

The Notch signaling pathway is fundamental to proper cardiovascular development and is now recognized as an important player in tumor angiogenesis. Two key Notch ligands have been implicated in tumor angiogenesis, Delta-like 4 and Jagged1. We introduce the proteins and how they work in normal developing vasculature and then discuss differing models describing the action of these Notch ligands in tumor angiogenesis. Endothelial Dll4 expression activates Notch resulting in restriction of new sprout development; for instance, in growing retinal vessels. In agreement with this activity, inhibition of Dll4-mediated Notch signaling in tumors results in hypersprouting of nonfunctional vasculature. This Dll4 inhibition may paradoxically lead to increased angiogenesis but poor tumor growth because the newly growing vessels are not functional. In contrast, Jagged1 has been described as a Notch ligand expressed in tumor cells that can have a positive influence on tumor angiogenesis, possibly by activating Notch on tumor endothelium. A novel Notch inhibitor, the Notch1 decoy, which blocks both Dll4 and Jagged1 has been recently shown to restrict tumor vessel growth. We discuss these models and speculate on therapeutic approaches.     

Tenascin-C regulates angiogenesis in tumor through the regulation of vascular endothelial growth factor expression

In order to verify whether tenascin-C (TN-C) is involved in angiogenesis as an extracellular signal molecule during tumorigenesis, cancerous cell transplantation experiments and coculture experiments were carried out, focusing on the regulation of vascular endothelial growth factor (VEGF). The A375 human melanoma cells introduced the GFP gene (A375-GFP), implanted subcutaneously into BALB/cA nude (WT) and TN-C knockout BALB/cA nude (TNKO) congenic mice. Furthermore, coculture experiments between A375-GFP and embryonic mesenchyme, which was prepared from both genotypes, were carried out to investigate the molecular mechanism in the cell-cell interactions. Both the content of TN-C and that of VEGF in the tumor and the conditioned medium were analyzed by the sandwich ELISA method. Seven days after transplantation of the A375-GFP, capillary nets became far more abundant in the tumors grown in WT mice than those in TNKO mice. Interestingly, VEGF and TN-C expressions showed antithetical expression patterns between the tumors in WT mice and those in TNKO mice. This peculiar phenomenon seems to be caused by a time lag prior to the onset of the mesenchymal regulation for the TN-C expression of A375-GFP. The coculture experiments revealed that WT mesenchyme had a much stronger effect than TNKO mesenchyme on both TN-C and VEGF expression. However, the defects of TNKO mesenchyme were restored in all cases by additional TN-C. These results clearly indicated that the expressions of both TN-C and VEGF depend on the surrounding mesenchyme, and that the function of mesenchyme is regulated by its own mesenchymal TN-C. In conclusion, the present data suggest that the matrix microenvironment organized by the host mesenchyme is very important for angiogenesis in tumor development.     

Targeting EZH2 regulates tumor growth and apoptosis through modulating mitochondria dependent cell-death pathway in HNSCC

EZH2 is a negative prognostic factor and is overexpressed or activated in most human cancers including head and neck squamous cell carcinoma (HNSCC). Analysis of The Cancer Genome Atlas (TCGA) HNSCC data indicated that EZH2 over-expression was associated with high tumor grade and conferred poor prognosis. EZH2 inhibition triggered cell apoptosis, cell cycle arrest and decreased cell growth in vitro. MICU1 (mitochondrial calcium uptake1) was shown to be down regulated when EZH2 expression was inhibited in HNSCC. When the EZH2 and MICU1 were inhibited, HNSCC cells became susceptible to cell cycle arrest and apoptosis. Mitochondrial membrane potential and cytosolic Ca²⁺ concentration analysis suggested that EZH2 and MICU1 were required to maintain mitochondrial membrane potential stability. A xenograft tumor model was used to confirm that EZH2 depletion inhibited HNSCC cell growth and induced tumor cell apoptosis. In summary, EZH2 is a potential anti-tumor target in HNSCC.     

Thrombospondin-1 type 1 repeat recombinant proteins inhibit tumor growth through transforming growth factor-beta-dependent and -independent mechanisms.

Thrombospondin-1 (TSP-1) is a potent inhibitor of tumor growth and angiogenesis. The antiangiogenic activity of TSP-1 has been mapped to the procollagen homology region and the type 1 repeats (TSR) using synthetic peptides. To elucidate the molecular mechanisms that are involved in the inhibition of tumor growth by the TSRs, we have expressed recombinant versions of these motifs and have assayed their ability to inhibit the growth of experimental B16F10 melanomas and Lewis lung carcinomas. Recombinant proteins that contain all three TSRs (3TSR) or the second TSR with (TSR2+RFK) or without (TSR2) the transforming growth factor-beta (TGFbeta) activating sequence (RFK) have been expressed in Drosophila S2 cells. In addition, recombinant proteins with mutations in either the RFK sequence (TSR2+QFK) or the WSHWSPW sequence [TSR2 (W/T)] of the second TSR have been prepared. Similar to platelet TSP-1, these proteins are potent inhibitors of endothelial cell migration, and 3TSR of human TSP-1 (3TSR/hTSP-1) and TSR2+RFK activate TGFbeta. An 81% inhibition of B16F10 tumor growth is observed at 2.5 mg (135 nmol)/kg/day of the recombinant 3TSR/hTSP-1. A comparable level of inhibition is observed with 2.5 mg (360 nmol)/kg/day of TSR2+RFK. By contrast, 3TSR of mouse TSP-2 (3TSR/mTSP-2), TSR2+QFK, and TSR2 are significantly less effective. TSR2+RFK and TSR2 reduce tumor vessel density, but TSR2+RFK has a greater effect on B16F10 tumor cell apoptosis and proliferation. Concurrent treatment of B16F10 tumor-bearing mice with TSR2+RFK and either a soluble form of the TGFbeta receptor or an antibody to active TGFbeta reduces the inhibition of B16F10 tumor growth to levels that are comparable with those of TSR2 and TSR2+QFK. By contrast, the presence of the TGFbeta-activating sequence does not increase the level of inhibition of Lewis lung carcinoma experimental tumor growth. These data indicate that the TSRs inhibit tumor growth by inhibition of angiogenesis and regulation of tumor cell growth and apoptosis. The regulation of tumor cell growth and apoptosis is TGFbeta dependent, whereas the inhibition of angiogenesis is not.     

Kallikrein 11 expressed in human breast cancer cells releases insulin-like growth factor through degradation of IGFBP-3

Expression of human kallikrein 11 (hK11), secreted as a trypsin-like serine protease, is related to the prognosis of some human cancers, but its physiological functions in the steps of cancer progression are still unknown. In order to elucidate the enzymatic function of hK11 we investigated the substrate of hK11 and expression of KLK11, the gene encoding hK11. Among 20 human cancer cell lines tested, two estrogen receptor possessing [ER(+)] breast cancers showed the highest expression of KLK11. The investigation of the hK11 substrate showed that hK11 could degrade itself and insulin-like growth factor (IGF) binding protein 3 (IGFBP-3). Expression of KLK11 was detected in both human breast cancer tissue and in non-cancerous mammary glands, and significantly higher KLK11 expression was observed in histological grade I/II than in grade III breast cancers. The above results indicate that the hK11 expressed in ER(+) breast cancer cells may play a crucial role in breast cancer progression by increasing the bioavailability of IGFs via degradation of IGFBP-3.     

Perhexiline promotes HER3 ablation through receptor internalization and inhibits tumor growth

Introduction

Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment.

Methods

We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform.

Results

We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo.

Conclusions

This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

Electronic supplementary material

The online version of this article (doi:10.1186/s13058-015-0528-9) contains supplementary material, which is available to authorized users.     

MiR-203 suppresses tumor growth and invasion and down-regulates MiR-21 expression through repressing Ran in esophageal cancer

The expression of miR-203 has been reported to be significantly down-regulated in esophageal cancer. We showed here that overexpression of miR-203 in esophageal cancer cells dramatically increased cell apoptosis and inhibited cell proliferation, migration and invasion as well as tumor growth and down-regulated miR-21 expression. We subsequently identified that small GTPase Ran was a target gene of miR-203. Furthermore, Ran restoration partially counteracted the tumor suppressive effects of miR-203 and increased miR-21 expression. Taken together, our findings suggest that miR-203 may act as novel tumor suppressor in esophageal cancer through down-regulating the expression of Ran and miR-21.     

IGFBP3 promotes esophageal cancer growth by suppressing oxidative stress in hypoxic tumor microenvironment

Insulin-like growth factor binding protein 3 (IGFBP3), a hypoxia-inducible gene, regulates a variety of cellular processes including cell proliferation, senescence, apoptosis and epithelial-mesenchymal transition (EMT). IGFBP3 has been linked to the pathogenesis of cancers. Most previous studies focus upon proapoptotic tumor suppressor activities of IGFBP3. Nevertheless, IGFBP3 is overexpressed in certain cancers including esophageal squamous cell carcinoma (ESCC), one of the most aggressive forms of squamous cell carcinomas (SCCs). The tumor-promoting activities of IGFBP3 remain poorly understood in part due to a lack of understanding as to how the tumor microenvironment may influence IGFBP3 expression and how IGFBP3 may in turn influence heterogeneous intratumoral cell populations. Here, we show that IGFBP3 overexpression is associated with poor postsurgical prognosis in ESCC patients. In xenograft transplantation models with genetically engineered ESCC cells, IGFBP3 contributes to tumor progression with a concurrent induction of a subset of tumor cells showing high expression of CD44 (CD44H), a major cell surface receptor for hyaluronic acid, implicated in invasion, metastasis and drug resistance. Our gain-of-function and loss-of-function experiments reveal that IGFBP3 mediates the induction of intratumoral CD44H cells. IGFBP3 cooperates with hypoxia to mediate the induction of CD44H cells by suppressing reactive oxygen species (ROS) in an insulin-like growth factor-independent fashion. Thus, our study sheds light on the growth stimulatory functions of IGFPB3 in cancer, gaining a novel mechanistic insight into the functional interplay between the tumor microenvironment and IGFBP3.     

Insulin-like growth factor-binding protein-1 (IGFBP-1) regulates human schwannoma proliferation, adhesion and survival

Merlin is a tumour suppressor involved in the development of a variety of tumours including mesotheliomas. Neurofibromatosis type 2 (NF2), a dominantly inherited tumour disease, is also caused by loss of merlin. NF2 patients suffer from multiple genetically well-defined tumours, schwannomas are most frequent among those. Using our in vitro model for human schwannoma, we found that schwannoma cells display enhanced proliferation because of the overexpression/activation of platelet-derived growth factor receptor and ErbB2/3, increased cell-matrix adhesion because of the overexpression of integrins, and decreased apoptosis. Mechanisms underlying schwannomas basal proliferation and cell-matrix adhesion are not understood. Here, we investigated insulin-like growth factor-binding protein-1 (IGFBP-1), which is expressed and released from central nervous system tumours and strongly overexpressed in schwannoma at the mRNA level. IGFBP-1 acts via β1-integrin and focal-adhesion-kinase (FAK), which are strongly overexpressed and basally activated in schwannoma. Using short hairpin RNA knockdown, small inhibitors and recombinant IGFBP-1, we demonstrate that schwannoma cells, in contrast to Schwann cells, release IGFBP-1 that activates the Src/FAK pathway, via integrin β1, potentiating schwannoma's proliferation and cell-matrix adhesion. We show that FAK localizes to the nucleus and Src triggers IGFBP-1 production. Further, we observed downregulation of the tumour-suppressor phosphatase and tensin homolog in schwannoma cells leading to increased activity of anti-apoptotic AKT. Thus, IGFBP-1/integrin β1/Src/FAK pathway has a crucial role in merlin-related tumourigenesis and therefore represents an important therapeutic target in the treatment of merlin-deficient tumours.     

EMMPRIN regulates tumor growth and metastasis by recruiting bone marrow-derived cells through paracrine signaling of SDF-1 and VEGF

EMMPRIN, a cell adhesion molecule highly expressed in a variety of tumors, is associated with poor prognosis in cancer patients. Mechanistically, EMMPRIN has been characterized to contribute to tumor development and progression by controlling the expression of MMPs and VEGF. In the present study, by using fluorescently labeled bone marrow-derived cells (BMDCs), we found that the down-regulation of EMMPRIN expression in cancer cells reduces tumor growth and metastasis, and is associated with the reduced recruitment of BMDCs. Further protein profiling studies suggest that EMMPRIN controls BMDC recruitment through regulating the secretion of soluble factors, notably, VEGF and SDF-1. We demonstrate that the expression and secretion of SDF-1 in tumor cells are regulated by EMMPRIN. This study reveals a novel mechanism by which EMMPRIN promotes tumor growth and metastasis by recruitment of BMDCs through controlling secretion and paracrine signaling of SDF-1 and VEGF.     

von Hippel-Lindau tumor suppressor protein regulates the assembly of intercellular junctions in renal cancer cells through hypoxia-inducible factor-independent mechanisms

Inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene is responsible for the development of renal cell cancers (RCC), pheochromocytomas, and tumors in other organs. The best known function of VHL protein (VHL) is to target the hypoxia-inducible factor (HIF) for proteasome degradation. VHL is also required for the establishment of an epithelial-like cell shape in otherwise fibroblastic-like RCC cell lines. However, the underlying mechanisms and whether this is linked to HIF remain undetermined. Because the breakage of intercellular junctions induces a fibroblastic-like phenotype in multiple cancer cell models, we hypothesized that VHL may be required for the assembly of intercellular junctions in RCC cells. Our experiments showed that VHL in RCC cell lines is necessary for the normal organization of adherens and tight intercellular junctions, the maintenance of cell polarity, and control of paracellular permeability. Additionally, 786-O cells reconstituted with wild-type VHL and with a constitutively active form of HIF-2alpha did not reproduce any of the phenotypic alterations of VHL-negative cells. In summary, we show that VHL inactivation in RCC cells disrupts intercellular junctions and cell shape through HIF-independent events, supporting the concept that VHL has additional functions beside its role in the regulation of HIF.     

VEGF-C-VEGFR3/Flt4 axis regulates mammary tumor growth and metastasis in an autocrine manner

Purpose: Lymphangiogenic factors, such as vascular endothelial growth factor-C (VEGF-C) and VEGFC-D, and their receptor, VEGF receptor-3 (VEGFR3), play a pivotal role in the promotion of metastasis to regional lymph nodes. In the present study we explored the role of VEGF-C as an autocrine growth factor for breast cancer cells. Methods: We examined the expression of VEGF-C and VEGFR3 in mammary tumor cells lines and examined whether blocking the VEGF-C-VEGFR3/Flt4 pathway using a VEGFR3 antagonist would inhibit proliferation of mammary tumor cells resulting in a decrease in tumor growth and metastasis. Results: We report expression of VEGF-C and its receptor VEGFR3 by mammary tumor cells, and their association with aggressiveness. Inhibition of VEGF-C-VEGFR3/Flt4 in mammary tumor cells decreased their proliferation and survival. Mammary tumor bearing mice treated with a VEGFR3 antagonist showed a significant decrease in tumor growth and the extent of spontaneous and experimental lung metastases. Conclusion: These findings demonstrate the VEGF-C-VEGFR3/Flt4 autocrine signaling pathway regulates mammary tumor cell survival and proliferation and that neutralization of VEGFR3 signaling might lead to development of a novel therapeutic approach for malignant breast cancer.