CD44 expressed on cancer-associated fibroblasts is a functional molecule supporting the stemness and drug resistance of malignant cancer cells in the tumor microenvironment.

Cells constituting the tumor microenvironment are attractive targets for developing new cancer therapies. Here we show that cancer-associated fibroblasts (CAFs) support tumor growth in vivo and maintain the stemness of cancer stem/initiating cells in an in vitro model using an established CAF cell line. We found that CD44 is abundantly expressed on CAFs. This molecule is a cancer stem cell marker in several tumors, but its role in tumorigenesis when expressed by CAFs has not been investigated. It is generally accepted that hypoxic and hyponutritional conditions are triggers of cancer malignancy. We found that CAFs strongly express CD44 in hypoxic and avascular areas in the tumor and that its expression on established CAFs is upregulated under hypoxic and hyponutritional conditions in vitro. In addition, CAF CD44-positivity in tumor tissues was increased after treatment with inhibitors of angiogenesis. Using cocultures and tumor sphere formation assays, CAFs from wild-type mice were found to sustain the stemness of cancer stem/initiating cells, while CD44-deficient CAFs did not. Furthermore, CD44 was involved in malignant cancer cell drug resistance mechanisms. In conclusion, our study suggests that CD44 on CAFs is a functional molecule contributing to the maintenance of cancer stem cell populations in the tumor microenvironment.

Possible role of mural cell-covered mature blood vessels in inducing drug resistance in cancer-initiating cells.

Cancer recurrence has been suggested to be induced by residual cancer-initiating cells (CICs) or cancer stem cells (CSCs) after chemotherapy. Moreover, it is possible that CICs/CSCs acquire more aggressive behavior after therapy as shown by invasion and metastasis. In the cancer microenvironment, CICs/CSCs may localize in a specific area, the so-called stem cell niche, and isolation of this niche is important to elucidate the molecular mechanism of how CICs/CSCs acquire malignancy. We analyzed whether CICs acquire drug resistance after cancer drug treatment in a tumor cell allograft model in which we could identify and isolate living CICs by detecting a higher level of transcriptional activity of the PSF1 gene promoter. In our models using Lewis lung carcinoma (LLC) mouse lung cancer and colon26 mouse colon cancer cell lines, we found that CICs in both tumors acquired drug resistance after cancer drug treatment. Interestingly, response to the anticancer drug was quite different between LLC and colon26 original tumors (ie, the proportion of CICs in LLC tumors increased but in colon26 tumors the proportion decreased). We found that CICs frequently localized near mature blood vessels in which endothelial cells were covered with mural cells and that the incidence of mature blood vessels in LLC tumors was four times higher than in colon26 tumors. These results suggest a relationship between mature blood vessels and CIC drug resistance.

Monoclonal antibody selectively recognizing murine but not human CD44.

Here we report on the generation of a monoclonal antibody (MAb) specific for murine, but not human CD44 obtained by immunization with cancer-associated fibroblasts. The monoclonal antibody MS44 recognizes CD44, as evidenced by immunoblotting of cell lysates. Additionally, MAb MS44 reacts with mouse but not human CD44 in flow cytometry. Thus, this antibody provides an effective tool to analyze CD44 from host (mouse) cells in human cancer cell xenograft models in mice.

Angiopoietin-1 alters tumor growth by stabilizing blood vessels or by promoting angiogenesis.

The maturation of blood vessels requires mural cell adhesion to endothelial cells. Angiopoietin-1 (Ang1), a ligand for Tie2 receptor expressed on endothelial cells, plays a critical role in cell adhesion between mural cells and endothelial cells and in endothelial cell sprouting from preexisting vessels in the absence of mural cells. Much information has been amassed on the Tie2-Ang1 system in physiological blood vessel formation during embryogenesis; however, the role of Ang1 in the tumor environment and its interaction with mural cells has not been well documented. Here we studied how Ang1 regulates maturation of blood vessels using the human colon cancer cell line HT29 and the human prostate cancer cell line PC3, and studied how Ang1 affects tumor growth. In a xenograft tumor model using female nude mice, we found that Ang1 enhanced angiogenesis and resulted in tumor growth in the case of PC3 tumors but suppressed tumor growth in the case of HT29 tumors. In PC3 tumors, the number of mural cells adhering to endothelial cells was less than that in HT29 tumors. Ang1 induced sprouting angiogenesis in PC3 tumors although there was little maturation of blood vessels. On the other hand, there was abundant mural cell adhesion to endothelial cells in HT29 tumors and Ang1 did not induce angiogenesis. These results suggest that Ang1 alters tumor growth in a manner that is dependent on the adhesion of mural cells and their localization in the tumor environment.

The carboxyl-terminal fragment of pro-HB-EGF reverses Bcl6-mediated gene repression.

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family, is synthesized as a type I transmembrane precursor (pro-HB-EGF). Ectodomain shedding of pro-HB-EGF yields an amino-terminal soluble ligand of EGF receptor (HB-EGF) and a carboxyl-terminal fragment (HB-EGF-CTF) consisting of the transmembrane and cytoplasmic domains. We previously showed that the HB-EGF-CTF translocates from the plasma membrane to the nucleus and plays a role as a signaling molecule. Immunoprecipitation showed that HB-EGF-CTF can associate with Bcl6, a transcriptional repressor in mammalian cells. A glutathione S-transferase pulldown assay revealed that HB-EGF-CTF interacted efficiently with zinc fingers 4-6 of Bcl6. A luciferase reporter assay showed that the nuclear translocation of HB-EGF-CTF following shedding reversed transcriptional repression of cyclin D2 by Bcl6. Additionally, the level of cyclin D2 protein increased and Bcl6 interaction with the cyclin D2 promoter decreased in parallel with the shedding of pro-HB-EGF at all endogenous levels. These findings suggest that HB-EGF-CTF is a potent regulator of gene expression via its interaction with the transcriptional repressor Bcl6.

Loss of HB-EGF in smooth muscle or endothelial cell lineages causes heart malformation.

Epidermal growth factor (EGF) and ErbB family molecules play a role in heart development and function. To investigate the role of EGF family member, heparin-binding EGF-like growth factor (HB-EGF) in heart development, smooth muscle and endothelial cell lineage-specific HB-EGF knockout mice were generated using the Cre/loxP system in combination with the SM22alpha or TIE2 promoter. HB-EGF knockout mice displayed enlarged heart valves, and over half of these mice died during the first postnatal week, while survivors showed cardiac hypertrophy. These results suggest that expression of HB-EGF in smooth muscle and/or endothelial cell lineages is essential for proper heart development and function in mice.

PLZF regulates Pbx1 transcription and Pbx1-HoxC8 complex leads to androgen-independent prostate cancer proliferation.

BACKGROUND: Promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor and negative regulator of the cell cycle, has been characterized as a prostatic androgen-responsive gene. DU145 cells show androgen-independent growth and lack PLZF gene expression. METHODS: We analyzed PLZF-regulating genes by DNA microarray using DU145 cells infected with LacZ- or PLZF-carrying adenoviruses. RESULTS: DNA microarray revealed that Pbx1 is a prominent suppressed gene in PLZF-overexpressing DU145 cells. Androgen receptor (AR)-expressing DU145 cells recovered androgen-dependent PLZF expression and subsequent repression of Pbx1 expression. Immunoprecipitation of Pbx1 in DU145 cells revealed a Pbx1-HoxC8 heterocomplex. siRNAs for Pbx1 and HoxC8 knocked downexpression of each, and this suppressed androgen-independent cell growth. Double knockdown of both Pbx1 and HoxC8 suppressed cell growth much more significantly. CONCLUSIONS: Androgen-independent cell line DU145 cells lack PLZF gene expression, resulting in the upregulation of Pbx1 and HoxC8 expression. The Pbx1-HoxC8 heterocomplex may lead to androgen-independent growth in prostate cancer.

Overexpression of ADAM9 enhances growth factor-mediated recycling of E-cadherin in human colon cancer cell line HT29 cells.

Growth factor-mediated stimulation of epithelial cells induces the disassembly of E-cadherin-mediated cell-cell adhesion. We found that overexpression of a disintegrin and metalloprotease 9 (ADAM9) enhanced growth factor-mediated induction of endocytosis and dynamic recycling of E-cadherin in HT29 human colon cancer cells. In addition, ubiquitination and degradation of E-cadherin were reduced in these cells. ADAM9 constitutively interacted with E-cadherin, and the two proteins co-localized at the plasma membrane of HT29 cells. Administration of a metalloprotease inhibitor or overexpression of an ADAM9 mutant lacking metalloprotease activity attenuated growth factor-dependent endocytosis and recycling of E-cadherin as well as scattering of HT29 cells. These results suggest that the metalloprotease activity of ADAM9 mediates growth factor-induced endocytosis and dynamic recycling of E-cadherin and prevents E-cadherin degradation.

Neuroglycan C, a novel member of the neuregulin family.

Neuroglycan C (NGC) is a transmembrane chondroitin sulfate proteoglycan expressed predominantly in the brain that possesses an EGF-like extracellular domain. The goal of the present study was to determine whether NGC may activate ErbB tyrosine kinases. A recombinant human NGC extracellular domain induced tyrosine phosphorylation of ErbB2 and ErbB3 as well as cell growth of the human breast tumor cell lines, T47D and MDA-MB-453. In vitro pull-down assay revealed that NGC could directly bind to a recombinant ErbB3-immunoglobulin Fc fusion protein (ErbB3-Fc) but not to ErbB1-Fc, ErbB2-Fc or ErbB4-Fc. A newly established anti-ErbB3 neutralizing monoclonal antibody (#5C3) almost completely blocked NGC-induced ErbB activation in MDA-MB-453 cells. Taken together, these data indicate that NGC is an active growth factor and a direct ligand for ErbB3 and that NGC transactivates ErbB2. Thus, NGC should be classified as the sixth member (neuregulin-6) of the neuregulin family.

The mechanism of cleavage of EGFR ligands induced by inflammatory cytokines in gastric cancer cells.

BACKGROUND & AIMS: The epidermal growth factor (EGF) receptor (EGFR) can be transactivated by many factors including G-protein-coupled receptor agonists and cytokines. Although this EGFR transactivation reportedly requires a disintegrin and metalloproteinase (ADAM) that sheds the ectodomain of EGFR ligands, the detailed mechanisms are still unknown. This study evaluated the mechanism of interleukin (IL)-8- and IL-1beta-dependent shedding of the EGFR ligand in KATO III cells. METHODS: We established transfectants stably expressing alkaline phosphatase-tagged heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor alpha, or amphiregulin precursors, and depleted ADAM proteins, using short interfering RNA against ADAM10, 12, or 17. We assessed shedding of EGFR ligands by measuring AP activities in the conditioned media after IL-1beta or IL-8 stimulation. EGFR activation was examined by immunoprecipitation and Western blotting using antiphosphotyrosine antibody. KB-R7785 and anti-IL-8 neutralizing antibody were used to inhibit activities of ADAMs and IL-8 action, respectively. RESULTS: IL-8 dose dependently released the EGFR ligands and transiently phosphorylated EGFR, with a peak at 15 minutes. KB-R7785 completely blocked IL-8-induced shedding and EGFR transactivation. Depletion of ADAM10 also dramatically reduced IL-8-induced shedding and EGFR transactivation, but depletion of ADAM12 and 17 did not. IL-1beta dose dependently enhanced shedding of HB-EGF, which was not blocked by KB-R7785 in the early phase. In the late phase, however, the EGFR transactivation was blocked by KB-R7785 and abrogated by anti-IL-8 neutralizing antibody. CONCLUSIONS: IL-8 induces shedding of EGFR ligands because of an ADAM10-dependent pathway in gastric cancer cells, whereas IL-1beta acts principally by an ADAM-independent pathway. IL-1beta-dependent prolonged EGFR transactivation involves multiple pathways, including an IL-8-dependent pathway.