Supplementary Table 1 from Extracellular Protease <i>ADAMTS9</i> Suppresses Esophageal and Nasopharyngeal Carcinoma Tumor Formation by Inhibiting Angiogenesis

Supplementary Methods from Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase 1act/Tubulin Interaction Is an Important Determinant of Mitotic Stability in Cultured HT1080 Human Fibrosarcoma Cells

Abstract Activation of the mitogen-activated protein kinase (MAPK) pathway plays a major role in neoplastic cell transformation. Using a proteomics approach, we identified α tubulin and β tubulin as proteins that interact with activated MAP/extracellular signal-regulated kinase kinase 1 (MEK1), a central MAPK regulatory kinase. Confocal analysis revealed spatiotemporal control of MEK1-tubulin colocalization that was most prominent in the mitotic spindle apparatus in variant HT1080 human fibrosarcoma cells. Peptide arrays identified the critical role of positively charged amino acids R108, R113, R160, and K157 on the surface of MEK1 for tubulin interaction. Overexpression of activated MEK1 caused defects in spindle arrangement, chromosome segregation, and ploidy. In contrast, chromosome polyploidy was reduced in the presence of an activated MEK1 mutant (R108A, R113A) that disrupted interactions with tubulin. Our findings indicate the importance of signaling by activated MEK1-tubulin in spindle organization and chromosomal instability. Cancer Res; 70(14); 6004–14. ©2010 AACR.

Supplementary Figures 1-3 from Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase 1act/Tubulin Interaction Is an Important Determinant of Mitotic Stability in Cultured HT1080 Human Fibrosarcoma Cells

Supplementary Figure 1 from Cancer Stem Cells Contribute to Cisplatin Resistance in Brca1/p53–Mediated Mouse Mammary Tumors

Supplementary Table 1 from Cancer Stem Cells Contribute to Cisplatin Resistance in <i>Brca1/p53</i>–Mediated Mouse Mammary Tumors

Supplementary Figure 1 from Cancer Stem Cells Contribute to Cisplatin Resistance in <i>Brca1/p53</i>–Mediated Mouse Mammary Tumors

Supplementary Figures S1-S3 from Monochromosome Transfer and Microarray Analysis Identify a Critical Tumor-Suppressive Region Mapping to Chromosome 13q14 and THSD1 in Esophageal Carcinoma

Supplementary Methods from Mitogen-Activated Protein/Extracellular Signal-Regulated Kinase Kinase 1^act/Tubulin Interaction Is an Important Determinant of Mitotic Stability in Cultured HT1080 Human Fibrosarcoma Cells

Newcastle disease virus (NDV) is a candidate agent for oncolytic virotherapy. Despite its potential, the exact mechanism of its oncolysis is still not known. Recently, we reported that NDV exhibited an increased oncolytic activity in hypoxic cancer cells. These types of cells negatively affect therapeutic outcome by overexpressing pro-survival genes under the control of the hypoxia-inducible factor (HIF). HIF-1 is a heterodimeric transcriptional factor consisting of a regulated α (HIF-1α) and a constitutive β subunit (HIF-1β). To investigate the effects of NDV infection on HIF-1α in cancer cells, the osteosarcoma (Saos-2), breast carcinoma (MCF-7), colon carcinoma (HCT116) and fibrosarcoma (HT1080) cell lines were used in the present study. Data obtained showed that a velogenic NDV infection diminished hypoxia-induced HIF-1α accumulation, leading to a decreased activation of its downstream target gene, carbonic anhydrase 9. This NDV-induced downregulation of HIF-1α occurred post-translationally and was partially abrogated by proteasomal inhibition. The process appeared to be independent of the tumour suppressor protein p53. These data revealed a correlation between NDV infection and HIF-1α downregulation, which highlights NDV as a promising agent to eliminate hypoxic cancer cells.

Purpose Newcastle disease virus (NDV) is an oncolytic virus that is known to have a higher preference to cancer cells than to normal cells. It has been proposed that this higher preference may be due to defects in the interferon (IFN) responses of cancer cells. The exact mechanism underlying this process, however, remains to be resolved. In the present study, we examined the antiviral response towards NDV infection of clear cell renal cell carcinoma (ccRCC) cells. ccRCC is associated with mutations of the von Hippel-Lindau tumor suppressor gene VHL , whose protein product is important for eliciting cellular responses to changes in oxygen levels. The most common first line treatment strategy of ccRCC includes IFN. Unfortunately, most ccRCC cases are diagnosed at a late stage and often are resistant to IFN-based therapies. Alternative treatment approaches, including virotherapy using oncolytic viruses, are currently being investigated. The present study was designed to investigate the mechanistic pathways underlying the response of ccRCC cells to oncolytic NDV infection. Methods and results We found that NDV induces activation of NF-κB in ccRCC cells by inducing phosphorylation and subsequent degradation of IκBα. IκBα was found to be phosphorylated as early as 1 hour post-infection and to result in rapid NF-κB nuclear translocation and activation. Importantly, p38 MAPK phosphorylation was found to occur upstream of the NDV-induced NF-κB activation. Restoration of VHL in ccRCC cells did not result in a reduction of this phosphorylation. A similar phenomenon was also observed in several other cancer-derived cell lines. Conclusion Our data provide evidence for involvement of the p38 MAPK/NF-κB/IκBα pathway in NDV infection and subsequent induction of apoptosis in ccRCC cells.

Bortezomib is the first proteasomal inhibitor (PI) to be used therapeutically for treating relapse cases of multiple myeloma and mantle cell lymphoma. A proposed mechanism for its action is that it prevents the proteasomal degradation of proapoptotic proteins, leading to enhanced apoptosis. Although the a subunit of hypoxia-inducible factor (HIF)-1 is not degraded with bortezomib treatment, the heterodimeric HIF-1 fails to transactivate target genes. HIP-1 and HIF-2 are related hypoxia-inducible transcription factors that are important for the survival of hypoxic tumor cells. The majority of reports have focused on the effects of bortezomib on the transcriptional activities of HIP-1, but not HIF-2. The present study investigated the effects of bortezomib on HIP-2 activity in cancer cells with different levels of HIP-1 alpha and HIP-2 alpha subunits. HIF-alpha subunit levels were detected using specific antibodies, while HIF transcriptional activities were evaluated using immunodetection, reverse transcription-polymerase chain reaction and luciferase reporter assay. Bortezomib treatment was found to suppress the transcription and expression of CA9, a HIP-1-specific target gene; however, it had minimal effects on EPO and GLUT-I, which are target genes of both HIP-1 and HIP-2. These data suggest that bortezomib attenuates the transcriptional activity only of HIF-1, and not HIP-2. This novel finding on the lack of an inhibitory effect of bortezomib on HIP-2 transcriptional activity has implications for the improvement of design and treatment modalities of bortezomib and other PI drugs.

Zinc-finger, MYND-type containing 10 (ZMYND10), or more commonly called BLU, expression is frequently downregulated in nasopharyngeal carcinoma (NPC) and many other tumors due to promoter hypermethylation. Functional evidence shows that the BLU gene inhibits tumor growth in animal assays, but the detailed molecular mechanism responsible for this is still not well understood. In current studies, we find that 93.5% of early-stage primary NPC tumors show downregulated BLU expression. Using a PCR array, overexpression of the BLU gene was correlated to the angiogenesis network in NPC cells. Moreover, expression changes of the MMP family, VEGF and TSP1, were often detected in different stages of NPC, suggesting the possibility that BLU may be directly involved in the microenvironment and anti-angiogenic activity in NPC development. Compared with vector-alone control cells, BLU stable transfectants, derived from poorly-differentiated NPC HONE1 cells, suppress VEGF165, VEGF189 and TSP1 expression at both the RNA and protein levels, and significantly reduce the secreted VEGF protein in these cells, reflecting an unknown regulatory mechanism mediated by the BLU gene in NPC. Cells expressing BLU inhibited cellular invasion, migration and tube formation. These in vitro results were further confirmed by in vivo tumor suppression and a matrigel plug angiogenesis assay in nude mice. Tube-forming ability was clearly inhibited, when the BLU gene is expressed in these cells. Up to 70-90% of injected tumor cells expressing increased exogenous BLU underwent cell death in animal assays. Overexpressed BLU only inhibited VEGF165 expression in differentiated squamous NPC HK1 cells, but also showed an anti-angiogenic effect in the animal assay, revealing a complicated mechanism regulating angiogenesis and the microenvironment in different NPC cell lines. Results of these studies indicate that alteration of BLU gene expression influences anti-angiogenesis pathways and is important for the development of NPC.