ag-490 and Colorectal-Neoplasms

Combination treatment with cetuximab and CPT-11 produces beneficial and synergistic effects in wild-type RAS metastatic colorectal cancer (mCRC) patients. However, the mechanism underlying this synergism is not yet understood. We examined whether cetuximab had a synergistic effect with CPT-11 and its active metabolite, SN38, and examined the molecular mechanism of the synergism between cetuximab and SN38 in CRC cells with various mutational status. We hypothesized that cetuximab promotes sensitivity to SN38 via suppression of heat shock protein 27 (HSP27), a protein involved in multidrug resistance through blocking the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway, which is associated with chemosensitivity. Four human CRC cell lines with different RAS and BRAF mutational status were used. Expression levels of HSP27 protein correlated with SN38 sensitivity in these cell lines (R=0.841, p=0.159). Exposure to cetuximab and various concentration of AG490, an inhibitor of JAK2, STAT3 and HSP27 protein levels, except in the KRAS G12V mutant line, SW620. A synergistic effect of cetuximab in combination with SN38 was observed in RAS and BRAF wild-type cells (here, Caco2), but not in the three other RAS- or BRAF-mutated cell lines. These results indicate that cetuximab may promote sensitivity to SN38 via suppression of HSP27 through blocking the JAK/STAT pathway in Caco2 cells. The mutational status of numerous downstream effectors, such as RAS and BRAF, is important in mono- or combination therapy with cetuximab. In conclusion, cetuximab may promote SN38 sensitivity via suppression of HSP27, through blocking the JAK/STAT signaling pathway, and shows synergistic effects when combined with SN38 in wild-type RAS CRC cells.

Topics: Antineoplastic Combined Chemotherapy Protocols; Camptothecin; Cell Line, Tumor; Cell Proliferation; Cetuximab; Colorectal Neoplasms; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation, Neoplastic; HSP27 Heat-Shock Proteins; Humans; Irinotecan; Janus Kinase 2; Mutation; Neoplasm Metastasis; Proto-Oncogene Proteins B-raf; Proto-Oncogene Proteins p21(ras); Signal Transduction; STAT3 Transcription Factor; Tyrphostins

Abnormalities in the JAK2/STAT3 pathway are involved in the pathogenesis of colorectal cancer (CRC), including apoptosis. However, the exact mechanism by which dysregulated JAK2/STAT3 signalling contributes to the apoptosis has not been clarified. To investigate the role of both JAK2 and STAT3 in the mechanism underlying CRC apoptosis, we inhibited JAK2 with AG490 and depleted STAT3 with a small interfering RNA. Our data showed that inhibition of JAK2/STAT3 signalling induced CRC cellular apoptosis via modulating the Bcl-2 gene family, promoting the loss of mitochondrial transmembrane potential (Δψm) and the increase of reactive oxygen species. In addition, our results demonstrated that the translocation of cytochrome c (Cyt c), caspase activation and cleavage of poly (ADP-ribose) polymerase (PARP) were present in apoptotic CRC cells after down-regulation of JAK2/STAT3 signalling. Moreover, inhibition of JAK2/STAT3 signalling suppressed CRC xenograft tumour growth. We found that JAK2/STAT3 target genes were decreased; meanwhile caspase cascade was activated in xenograft tumours. Our findings illustrated the biological significance of JAK2/STAT3 signalling in CRC apoptosis, and provided novel evidence that inhibition of JAK2/STAT3 induced apoptosis via the mitochondrial apoptotic pathway. Therefore, JAK2/STAT3 signalling may be a potential target for therapy of CRC.

Topics: Animals; Apoptosis; Caspases; Cell Line, Tumor; Colorectal Neoplasms; Cytochromes c; Cytosol; Gene Expression Regulation, Neoplastic; Humans; Janus Kinase 2; Male; Membrane Potential, Mitochondrial; Mice; Mice, Nude; Mitochondria; Models, Biological; Poly(ADP-ribose) Polymerases; Protein Transport; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Xenograft Model Antitumor Assays

Abnormalities in the STAT3 pathway are involved in the oncogenesis of several cancers. However, the mechanism by which dysregulated STAT3 signaling contributes to the progression of human colorectal cancer (CRC) has not been elucidated, nor has the role of JAK, the physiological activator of STAT3, been evaluated. To investigate the role of both JAK and STAT3 in CRC progression, we inhibited JAK with AG490 and depleted STAT3 with a SiRNA. Our results demonstrate that STAT3 and both JAK1 and 2 are involved in CRC cell growth, survival, invasion, and migration through regulation of gene expression, such as Bcl-2, p1(6ink4a), p21(waf1/cip1), p27(kip1), E-cadherin, VEGF, and MMPs. Importantly, the FAK is not required for STAT3-mediated regulation, but does function downstream of JAK. In addition, our data show that proteasome-mediated proteolysis promotes dephosphorylation of the JAK2, and consequently, negatively regulates STAT3 signaling in CRC. Moreover, immunohistochemical staining reveals that nuclear staining of phospho-STAT3 mostly presents in adenomas and adenocarcinomas, and a positive correlation is found between phospho-JAK2 immunoreactivity and the differentiation of colorectal adenocarcinomas. Therefore, our findings illustrate the biologic significance of JAK1, 2/STAT3 signaling in CRC progression and provide novel evidence that the JAK/STAT3 pathway may be a new potential target for therapy of CRC.

Topics: Apoptosis; Cadherins; Cell Cycle; Colorectal Neoplasms; Cysteine Proteinase Inhibitors; Down-Regulation; Focal Adhesion Kinase 1; Humans; Janus Kinase 1; Janus Kinase 2; Leupeptins; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Protein Kinase Inhibitors; RNA, Small Interfering; Signal Transduction; STAT3 Transcription Factor; Tyrphostins; Vascular Endothelial Growth Factor A

Nuclear accumulation of beta-catenin is a key event for the development of colorectal cancer. Little is known, however, about the mechanisms underlying translocation of beta-catenin from the cytoplasm or the membrane to the nucleus. The present study examined whether signal transducers and activators of transcription 3 (STAT3) activation is involved in the nuclear accumulation of beta-catenin in colorectal cancer cells. Of the 90 primary colorectal cancer tissues, 40 (44.4%) were positive for nuclear staining of p-STAT3 and 63 (70.0%) were positive for nuclear staining of beta-catenin. The nuclear staining of both p-STAT3 and beta-catenin were observed predominantly in the periphery of the cancer tissues. Importantly, of the 40 tumors with p-STAT3 nuclear staining, 37 (92.5%) were also positive for nuclear beta-catenin staining and there was a significant correlation between p-STAT3 and beta-catenin nuclear staining (P < 0.01). Coexpression of nuclear p-STAT3 and beta-catenin was associated with lower patient survival (P < 0.01). In an in vitro study using a human colon cancer cell line, SW480, inhibition of STAT3 by dominant negative STAT3 or the Janus kinase inhibitor, AG490, induced translocation of beta-catenin from the nucleus to the cytoplasm or membrane. Luciferase assays revealed that STAT3 inhibition resulted in significant suppression of beta-catenin/T-cell factor transcription in association with significant inhibition of cell proliferation (P < 0.05). These findings suggest that in colorectal cancer, STAT3 activation is involved in the nuclear accumulation of beta-catenin, resulting in poor patient survival.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents; beta Catenin; Cell Growth Processes; Cell Nucleus; Colorectal Neoplasms; Female; Humans; Immunohistochemistry; Lymphatic Metastasis; Male; Middle Aged; STAT3 Transcription Factor; Transcription, Genetic; Transfection; Tumor Cells, Cultured; Tyrphostins