Takeshi Kawano

Direct targeting of the mucin 1 oncoprotein blocks survival and tumorigenicity of human breast carcinoma cells.

The mucin 1 (MUC1) oncoprotein is aberrantly overexpressed by approximately 90% of human breast cancers. However, there are no effective agents that directly inhibit MUC1 and induce death of breast cancer cells. We have synthesized a MUC1 inhibitor (called GO-201) that binds to the MUC1 cytoplasmic domain and blocks the formation of MUC1 oligomers in cells. GO-201, and not an altered version, attenuates targeting of MUC1 to the nucleus of human breast cancer cells, disrupts redox balance, and activates the DNA damage response. GO-201 also arrests growth and induces necrotic death. By contrast, the MUC1 inhibitor has no effect on cells null for MUC1 expression or nonmalignant mammary epithelial cells. Administration of GO-201 to nude mice bearing human breast tumor xenografts was associated with loss of tumorigenicity and extensive necrosis, which results in prolonged regression of tumor growth. These findings show that targeting the MUC1 oncoprotein is effective in inducing death of human breast cancer cells in vitro and in tumor models.

Telomerase overexpression in K562 leukemia cells protects against apoptosis by serum deprivation and double-stranded DNA break inducing agents, but not against DNA synthesis inhibitors

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusions. The maintenance of telomeres is essential for chromosomal stability. Telomerase is regulated by human telomerase reverse transcriptase (hTERT). c-Myc oncoprotein is also implicated in the positive regulation of hTERT expression. We show here that two clones of hTERT-transfected K562 erythroleukemia cells have elongated telomeres (22.5 and 24.0 kb), whereas telomere length of both c-Myc-transfected K562 cells and parental K562 cells is 6.5 kb. Telomerase activity and hTERT mRNA expression increased in hTERT-transfected K562 cells, while the expression levels of telomerase activity and hTERT in c-Myc-transfected K562 cells were similar to that in parental K562 cells, despite an overexpression of c-Myc. Importantly, we found that hTERT-transfected K562 cells are protected against apoptosis induced by serum deprivation and double-stranded DNA break inducing agents (ionizing irradiation, and etoposide (VP-16)), but not against DNA synthesis inhibitors (1-beta-D-arabinofuranosylcytosine and hydroxyurea). These findings suggest that overexpression of telomerase by transfecting hTERT confers telomere-elongation and resistance to double-stranded DNA break inducing agents.

MUC1-C Oncoprotein Functions as a Direct Activator of the Nuclear Factor-κB p65 Transcription Factor

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies. The mucin 1 (MUC1) oncoprotein is overexpressed in human carcinomas and, like NF-kappaB, blocks cell death and induces transformation. The present studies show that MUC1 constitutively associates with NF-kappaB p65 in carcinoma cells. The MUC1 COOH-terminal subunit (MUC1-C) cytoplasmic domain binds directly to NF-kappaB p65 and, importantly, blocks the interaction between NF-kappaB p65 and its inhibitor IkappaBalpha. We show that NF-kappaB p65 and MUC1-C constitutively occupy the promoter of the Bcl-xL gene in carcinoma cells and that MUC1-C contributes to NF-kappaB-mediated transcriptional activation. Studies in nonmalignant epithelial cells show that MUC1-C interacts with NF-kappaB in the response to tumor necrosis factor-alpha stimulation. Moreover, tumor necrosis factor-alpha induces the recruitment of NF-kappaB p65-MUC1-C complexes to NF-kappaB target genes, including the promoter of the MUC1 gene itself. We also show that an inhibitor of MUC1-C oligomerization blocks the interaction with NF-kappaB p65 in vitro and in cells. The MUC1-C inhibitor decreases MUC1-C and NF-kappaB p65 promoter occupancy and expression of NF-kappaB target genes. These findings indicate that MUC1-C is a direct activator of NF-kappaB p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-kappaB pathway.

MUC1 Oncoprotein Blocks Death Receptor–Mediated Apoptosis by Inhibiting Recruitment of Caspase-8

Stimulation of the death receptor superfamily induces the activation of caspase-8 and thereby the apoptotic response. The MUC1 oncoprotein is aberrantly overexpressed by diverse human malignancies and inhibits stress-induced apoptosis. The present results show that MUC1 blocks activation of caspase-8 and apoptosis in the response of malignant cells to tumor necrosis factor alpha, tumor necrosis factor-related apoptosis-inducing ligand, and Fas ligand. The results show that MUC1 associates constitutively with caspase-8. The MUC1 cytoplasmic domain (MUC1-CD) binds directly to the caspase-8 p18 fragment upstream to the catalytic Cys(360) site. The results also show that MUC1-CD binds to Fas-associated death domain (FADD) at the death effector domain. In nonmalignant epithelial cells, MUC1 interacts with caspase-8 and FADD as an induced response to death receptor stimulation. The functional significance of these interactions is supported by the demonstration that MUC1 competes with caspase-8 for binding to FADD and blocks recruitment of caspase-8 to the death-inducing signaling complex. These findings indicate that MUC1 is of importance to the physiologic regulation of caspase-8 activity and that overexpression of MUC1, as found in human malignancies, could contribute to constitutive inhibition of death receptor signaling pathways.

MUC1-C Oncoprotein Promotes STAT3 Activation in an Autoinductive Regulatory Loop

An inflammatory response of epithelial cells may be co-opted to promote cancer cell survival. Caught in a Loop Mucin 1 (MUC1), a glycoprotein found at the apical surface of epithelial cells, is overexpressed in various carcinomas, including breast cancer; indeed, its overexpression can elicit cell transformation. The MUC1 carboxyl-terminal receptor subunit (MUC1-C) has been implicated in several signaling pathways, and here Ahmad et al. link it to signaling downstream of interleukin-6 (IL-6) and other inflammatory cytokines. They found that MUC1-C associated with components of the IL-6 receptor complex in breast cancer cells, in which it was required for JAK1-mediated phosphorylation of STAT3, and promoted STAT3 binding to and activation of target genes (including both MUC1 and STAT3). IL-6 stimulated a less prominent basal interaction between MUC1-C and STAT3 in nonmalignant breast epithelial cells. The authors thus propose that MUC1-C, by promoting activation of STAT3-dependent genes, may play a protective role in the inflammatory response of breast epithelial cells and that this response gets locked into an autoinductive loop in cancer cells, thereby promoting their resistance to cell death. Signal transducer and activator of transcription 3 (STAT3) is activated in human breast cancer and other malignancies. Mucin 1 (MUC1) is a heterodimeric cell surface glycoprotein that is overexpressed in human carcinomas and, like STAT3, promotes cell survival and induces transformation. We found that in breast cancer cells, the MUC1 carboxyl-terminal receptor subunit (MUC1-C) associates with the gp130–Janus-activated kinase 1 (JAK1)–STAT3 complex. The MUC1-C cytoplasmic domain interacted directly with JAK1 and STAT3, and MUC1-C was necessary for JAK1-mediated STAT3 activation. In turn, MUC1-C and activated STAT3 occupied the promoter of MUC1, and MUC1-C contributed to STAT3-mediated activation of MUC1 transcription. The MUC1-C inhibitor GO-201 blocked the MUC1-C interaction with STAT3, thereby decreasing MUC1-C and STAT3 occupancy on the MUC1 and STAT3 promoters and activation of STAT3 target genes, including MUC1 itself. These findings indicate that MUC1-C promotes STAT3 activation and that MUC1-C and STAT3 function in an autoinductive loop that may play a role in cancer cell survival.

MUC1-C oncoprotein functions as a direct activator of the nuclear factor-kappaB p65 transcription factor.

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies. The mucin 1 (MUC1) oncoprotein is overexpressed in human carcinomas and, like NF-kappaB, blocks cell death and induces transformation. The present studies show that MUC1 constitutively associates with NF-kappaB p65 in carcinoma cells. The MUC1 COOH-terminal subunit (MUC1-C) cytoplasmic domain binds directly to NF-kappaB p65 and, importantly, blocks the interaction between NF-kappaB p65 and its inhibitor IkappaBalpha. We show that NF-kappaB p65 and MUC1-C constitutively occupy the promoter of the Bcl-xL gene in carcinoma cells and that MUC1-C contributes to NF-kappaB-mediated transcriptional activation. Studies in nonmalignant epithelial cells show that MUC1-C interacts with NF-kappaB in the response to tumor necrosis factor-alpha stimulation. Moreover, tumor necrosis factor-alpha induces the recruitment of NF-kappaB p65-MUC1-C complexes to NF-kappaB target genes, including the promoter of the MUC1 gene itself. We also show that an inhibitor of MUC1-C oligomerization blocks the interaction with NF-kappaB p65 in vitro and in cells. The MUC1-C inhibitor decreases MUC1-C and NF-kappaB p65 promoter occupancy and expression of NF-kappaB target genes. These findings indicate that MUC1-C is a direct activator of NF-kappaB p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-kappaB pathway.

MUC1-C ONCOPROTEIN FUNCTIONS AS A DIRECT ACTIVATOR OF THE NF-κB p65 TRANSCRIPTION FACTOR

Nuclear factor-kappaB (NF-kappaB) is constitutively activated in diverse human malignancies. The mucin 1 (MUC1) oncoprotein is overexpressed in human carcinomas and, like NF-kappaB, blocks cell death and induces transformation. The present studies show that MUC1 constitutively associates with NF-kappaB p65 in carcinoma cells. The MUC1 COOH-terminal subunit (MUC1-C) cytoplasmic domain binds directly to NF-kappaB p65 and, importantly, blocks the interaction between NF-kappaB p65 and its inhibitor IkappaBalpha. We show that NF-kappaB p65 and MUC1-C constitutively occupy the promoter of the Bcl-xL gene in carcinoma cells and that MUC1-C contributes to NF-kappaB-mediated transcriptional activation. Studies in nonmalignant epithelial cells show that MUC1-C interacts with NF-kappaB in the response to tumor necrosis factor-alpha stimulation. Moreover, tumor necrosis factor-alpha induces the recruitment of NF-kappaB p65-MUC1-C complexes to NF-kappaB target genes, including the promoter of the MUC1 gene itself. We also show that an inhibitor of MUC1-C oligomerization blocks the interaction with NF-kappaB p65 in vitro and in cells. The MUC1-C inhibitor decreases MUC1-C and NF-kappaB p65 promoter occupancy and expression of NF-kappaB target genes. These findings indicate that MUC1-C is a direct activator of NF-kappaB p65 and that an inhibitor of MUC1 function is effective in blocking activation of the NF-kappaB pathway.

MUC1 Oncoprotein Regulates Bcr-Abl Stability and Pathogenesis in Chronic Myelogenous Leukemia Cells

Chronic myelogenous leukemia (CML) results from expression of the Bcr-Abl fusion protein in hematopoietic stem cells. The MUC1 heterodimeric protein is aberrantly overexpressed in diverse human carcinomas. The present studies show that MUC1 is expressed in the human K562 and KU812 CML cell lines. The results show that MUC1 associates with Bcr-Abl through a direct interaction between the Bcr N-terminal region and the MUC1 cytoplasmic domain. Stable silencing of MUC1 decreased cytoplasmic Bcr-Abl levels by promoting Bcr-Abl degradation. Silencing MUC1 was also associated with decreases in K562 and KU812 cell self-renewal capacity and with a more differentiated erythroid phenotype. The results further show that silencing MUC1 increases sensitivity of CML cells to imatinib-induced apoptosis. Analysis of primary CML blasts confirmed that, as found with the CML cell lines, MUC1 blocks differentiation and the apoptotic response to imatinib treatment. These findings indicate that MUC1 stabilizes Bcr-Abl and contributes to the pathogenesis of CML cells by promoting self renewal and inhibiting differentiation and apoptosis.

MUC1 induces metastasis in esophageal squamous cell carcinoma by upregulating matrix metalloproteinase 13

Esophagus squamous cell carcinoma (ESCC) is one of the most deadly malignances because of its high frequency of metastasis. Given the associations of MUC1 with ESCC and tumor metastasis, we explored a potential role of MUC1 in ESCC metastasis. Among 40 ESCC and 20 paired normal tissue specimens examined, we found a significant increase of MUC1 expression in ESCC and more importantly, that expression of MUC1 and MMP13 are strongly correlated in patients who had lymph node metastasis. Studies with cell models indicated that overexpression of MUC1 upregulates the expression of MMP13, leading to increased cell migration. In support of a mode of transcriptional regulation, promoter analysis revealed that MUC1 stimulates MMP13 expression through the Runx-2-binding site. The link of MUC1 to cell motility was further confirmed by the finding that depletion of MUC1 resulted in reduced expression of MMP13 and cell migration, invasion and adhesion. Moreover, the loss of cell metastatic potential was rescued by overexpression of MMP13 completely. Collectively, our findings indicate that MUC1 contributes to ESCC metastasis by stimulating MMP13 expression, suggesting MUC1 as a novel diagnostic biomarker and therapeutic target in ESCC.

Antitumor activity of TMPyP4 interacting G-quadruplex in retinoblastoma cell lines

To investigate the molecular mechanism of the antitumor activity of the cationic porphyrin 5, 10, 15, 20-tetra-(N-methyl-4-pyridyl)porphyrin (TMPyP4) in retinoblastoma cell lines, Y79 and WERI-Rb1 cells were treated with TMPyP4 for 0-72 h, after which growth inhibition, modulation of the cell cycle and the induction of apoptosis were examined. In addition, the effect of TMPyP4 on the susceptibility to irradiation was evaluated in Y79 and WERI-Rb1 cells. In vitro telomeric repeat amplification protocol assay showed TMPyP4 (10-100 microM) directly blocked telomerase elongation, suggesting that TMPyP4 can form stable guanine (G)-quadruplexes in extending telomere repeats in substrate oligonucleotides. The antiproliferative activities of TMPyP4 assessed with the MTS assay and expressed in terms of IC(50): Y79 cells, 60 microM; WERI-Rb1 cells, 45 microM. Treatment with TMPyP4 at doses of 10, 20, 50 or 100 microM for 48 or 72 h significantly inhibited the growth of Y79 and WERI-Rb1 cells. Apoptosis, as assessed with CaspACE FITC-VAD-FMK, was induced by TMPyP4 in a dose-dependent manner. Induction of apoptosis by TMPyP4 was associated with increased expression of phosphorylated DNA damage response factor H2AX (Ser139), phosphorylated p53 (Ser46) protein and activation of mitogen-activated protein kinases in Y79 and WERI-Rb1 cells. Moreover, TMPyP4 significantly enhanced the susceptibility to irradiation in both cell lines. This study provides insight into the molecular mechanism of the antitumor effects of TMPyP4. G-quadruplex structure may be a potential therapeutic target in retinoblastoma.