Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of wh... more Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)-the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib, caused by elevated expression of components of the β-catenin pathway, exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in these gefitinib-resistant cancer cells. Moreover, overexpression of MTHFD2 in gefitinibsensitive lung cancer cells conferred resistance to gefitinib. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.
Cancer stem cells are thought to be responsible for tumor growth, recurrence, and resistance to c... more Cancer stem cells are thought to be responsible for tumor growth, recurrence, and resistance to conventional cancer therapy. However, it is still unclear how they are maintained in tumor tissues. Here, we show that the growth differentiation factor 15 (GDF15), a member of the TGFβ family, may maintain cancer stem-like cells in breast cancer tissues by inducing its own expression in an autocrine/paracrine manner. We found that GDF15, but not TGFβ, increased tumor sphere formation in several breast cancer cell lines and patient-derived primary breast cancer cells. As expected, TGFβ strongly stimulated the phosphorylation of Smad2. GDF15 also stimulated the phosphorylation of Smad2, but the GDF15-induced tumor sphere forming efficiency was not significantly affected by treatment with SB431542, an inhibitor of the TGFβ signaling. Although TGFβ transiently activated ERK1/2, GDF15 induced prolonged activation of ERK1/2. Treatment with U0126, an inhibitor of the MEK-ERK1/2 signaling, great...
Tumor cells respond to their microenvironment, which can include hypoxia and malnutrition, and ad... more Tumor cells respond to their microenvironment, which can include hypoxia and malnutrition, and adapt their metabolism to survive and grow. Some oncogenes are associated with cancer metabolism via regulation of the related enzymes or transporters. However, the importance of metabolism and precise metabolic effects of oncogenes in colorectal cancer remain unclear. We found that colorectal cancer cells survived under the condition of glucose depletion, and their resistance to such conditions depended on genomic alterations rather than on KRAS mutation alone. Metabolomic analysis demonstrated that those cells maintained tricarboxylic acid cycle activity and ATP production under such conditions. Furthermore, we identified pivotal roles of GLUD1 and SLC25A13 in nutritional stress. GLUD1 and SLC25A13 were associated with tumor aggressiveness and poorer prognosis of colorectal cancer. In conclusion, GLUD1 and SLC25A13 may serve as new targets in treating refractory colorectal cancer which s...
In vivo studies: Sprague-Dawley rats, 300-400 g, were anaesthetised with α-chloralose and ketamin... more In vivo studies: Sprague-Dawley rats, 300-400 g, were anaesthetised with α-chloralose and ketamine and colonic intraluminal pressure and blood pressure were recorded. In other experiments, visceromotor responses to colorectal distension (VMR) were measured by recording the EMG from abdominal muscle. Fecal output was measured from conscious rats housed in a quiet environment. Immunohistochemistry was done on spinal cord sections and receptor and peptide gene expression were determined by RT-PCR. Results Receptor immunoreactivity occurred in the cell bodies of autonomic pre-ganglionic neurons at all levels of the spinal cord, from T1 to S2. RT-PCR from all levels of the cord detected transcripts for both the ghrelin receptor and ghrelin itself. Intravenous infusion of CP-464709 (5-20 mg/kg/h) caused propulsive contractions of the colon that was associated with increased propulsion of colonic contents in anaesthetised rats, as well as a blood pressure rise. The contractions induced by the agonist were large and regular, but it had no direct effect on the muscle. In conscious rats, subcutaneous CP464709 caused fecal expulsion. Intrathecal application of CP464709 or ghrelin at L6-S1, the region of defecation control centres, but not application at ponto-medullary levels, elicited propulsive contractions. The agonists administered to the thoracic cord raised blood pressure by 40 mm Hg. The colonic motility evoked by intravenous CP464709 was prevented if the pelvic nerve outflows were severed. When high dose of CP464709, applied intrathecally, was used to desensitise its receptors, the effect of intravenous CP464709 was blocked. CP464709 did not affect small intestine motility or the amplitudes of visceromotor reflexes caused by colorectal distension. Conclusions Activation of ghrelin receptors in the lumbo-sacral spinal cord triggers coordinated propulsive contractions that empty the colon. The pathways through which these responses are generated pass out of the spinal cord via the pelvic nerves and cause propulsive contractions through activation of enteric neurons. Activation of ghrelin receptors in thoracic and lumbo-sacral regions increases blood pressure.
From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently ... more From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3 clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth facto...
The fibroblast growth factor receptor substrate (FRS)-2 protein family comprises FRS2a, a wellkno... more The fibroblast growth factor receptor substrate (FRS)-2 protein family comprises FRS2a, a wellknown central mediator for fibroblast growth factor signaling, and FRS2b, whose endogenous expression pattern and function are not yet defined. Immunohistochemical analysis revealed that expression of FRS2b was restricted to neural tissues and it colocalized with Tuj1, a neuronal marker. There are two distinct patterns of FRS2b expression in neural cells: punctate and cup/ring-shaped; moreover, some particles colocalized with lysosomes. Stimulation with brain-derived neurotrophic factor (BDNF) enhanced FRS2b phosphorylation and the cup/ring-shaped pattern. These results suggest a probable role of FRS2b in the intracellular degradation systems of neural cells, which involves lysosomes.
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 1994
Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a liga... more Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with the capacity of the
The CD74-Neuregulin1 (NRG1) fusion gene was recently identified as novel driver of invasive mucin... more The CD74-Neuregulin1 (NRG1) fusion gene was recently identified as novel driver of invasive mucinous adenocarcinoma, a malignant form of lung cancer. However, the function of the CD74-NRG1 fusion gene in adenocarcinoma pathogenesis and the mechanisms by which it may impart protumorigenic characteristics to cancer stem cells (CSC) is still unclear. In this study, we found that the expression of the CD74-NRG1 fusion gene increased the population of lung cancer cells with CSC-like properties. CD74-NRG1 expression facilitated sphere formation not only of cancer cells, but also of nonmalignant lung epithelial cells. Using a limiting dilution assay in a xenograft model, we further show that the CD74-NRG1 fusion gene enhanced tumor initiation. Mechanistically, we found that CD74-NRG1 expression promoted the phosphorylation of ErbB2/3 and activated the PI3K/Akt/NF-κB signaling pathway. Furthermore, the expression of the secreted insulin-like growth factor 2 (IGF2) and phosphorylation of its...
Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer and is the most common and f... more Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer and is the most common and fatal cancer worldwide. Specific tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR), such as gefitinib, have been effective in some NSCLC patients and are being used in the clinical setting as pioneer molecularly targeted cancer drugs. However, many patients have not responded to these drugs, and have acquired resistance after long-term treatment. To identify other potential NSCLC molecular targets, we used DNA microarrays to examine gene expression profiles of gefitinib-resistant PC9/ZD cells that are derived from gefitinib-sensitive PC9 cells and harbor a threonine to methionine mutation at codon 790 (T790M) in EGFR, a known mechanism of acquired resistance to gefitinib. We found that N-cadherin expression was significantly upregulated in PC9/ZD cells compared with PC9 cells. Inhibition of N-cadherin expression by siRNA or treatment with antibodies against N-cadherin induced apoptosis of PC9/ZD cells in association with reduced phosphorylation of Akt and Bad, a proapoptotic protein. Moreover, inhibition of Akt expression by siRNA or treatment with an inhibitor for phosphatidylinositol (PI)-3 kinase reduced survival of PC9/ZD cells. In addition, we found several N-cadherin-expressing lung cancer cells that showed inherent resistance to gefitinib treatment and reduced survival owing to siRNA-induced inhibition of N-cadherin expression. Thus, it appears that N-cadherin maintains the survival of the gefitinib-resistant lung cancer cells via the PI-3 kinase/Akt survival pathway. From these results, we propose that N-cadherin signaling contributes, at least in part, to the survival mechanisms of gefitinib-resistant NSCLC cells and that N-cadherin is a potential molecular target in the treatment of NSCLC.
Interleukin 3 (IL-3) not only induces DNA synthesis of haematopoietic cells but also maintains th... more Interleukin 3 (IL-3) not only induces DNA synthesis of haematopoietic cells but also maintains their viability by suppressing apoptosis. IL-3 stimulates tyrosine phosphorylation of the Shc adaptor protein and thereby formation of a complex of Shc with Grb2 at phosphorylated tyrosine (Y) residue 317-Shc. This pathway is implicated in Ras/mitogen-activated protein kinase (MAPK) activation towards c-fos gene expression. We examined the possible involvement of Shc in the antiapoptotic activity of IL-3. Conditional overexpression of the Shc SH2 domain, a dominant-negative mutant of Shc, was found to induce apoptosis of IL-3-dependent BaIF3 cells along with a reduction of c-myc gene expression. Apoptosis was rescued by the exogenously introduced c-myc gene. Since we identify novel tyrosine phosphorylation sites of Shc: Y239 and Y240, their role on cell survival was tested by mutational analysis. Ba/F3 cells expressing mutant Shc Y317F, which is unable to stimulate efficiently the Ras pathway, still showed resistance to apoptosis. However, cells expressing Shc Y239/240F, which is able to stimulate the Ras pathway, were sensitive to apoptosis. In these cells, induction of the c-myc gene was reduced. These findings suggest that a new signalling pathway for cell survival is generated from Y239/240 of Shc to the nuclei involving c-myc gene expression.
Unlike other cyclins that positively regulate the cell cycle, cyclin G2 (CCNG2) regulates cell pr... more Unlike other cyclins that positively regulate the cell cycle, cyclin G2 (CCNG2) regulates cell proliferation as a tumor suppressor gene. A decreased CCNG2 expression serves as a marker for poor prognosis in several types of cancer. The aim of the present study was to clarify the correlation of CCNG2 expression with overall survival and histopathological factors in pancreatic cancer patients. This retrospective analysis included data from 36 consecutive patients who underwent complete surgical resection for pancreatic cancer and did not undergo any preoperative therapies. The association between prognoses and the expression of CCNG2 was assessed using immunohistochemical staining. Multivariate analysis identified that the expression of CCNG2 is an independent prognostic factor. In addition, the Kaplan-Meier curve for overall survival revealed that decreased expression of CCNG2 was a consistent indicator of poor prognosis in pancreatic cancer patients (P=0.0198). A decreased CCNG2 expression significantly correlated with venous invasion in tumor specimens and the tumor invasion depth. In conclusion, CCNG2 expression inversely reflected cancer progression and may be a novel, independent prognostic marker in pancreatic cancer.
CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Weste... more CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Western blotting with CRK SH3 has demonstrated that it binds to 135-to 145-, 160-, and 180-kDa proteins. The 135to 145-kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange protein. Here, we report on the molecular cloning of the 180-kDa protein, which is designated DOCK180 (180-kDa protein downstream of CRK). The isolated cDNA contains a 5,598-bp open reading frame encoding an 1,866-amino-acid protein. The deduced amino acid sequence did not reveal any significant homology to known proteins, except that an SH3 domain was identified at its amino terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulated diffusely in the cytoplasm and did not have any effect on cell morphology, farnesylated DOCK180 was localized on the cytoplasmic membrane and changed spindle 3T3 cells to flat, polygonal cells. These results suggest that DOCK180 is a new effector molecule which transduces signals from tyrosine kinases through the CRK adaptor protein.
Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of wh... more Tumor recurrence is attributable to cancer stem-like cells (CSCs), the metabolic mechanisms of which currently remain obscure. Here, we uncovered the critical role of folate-mediated one-carbon (1C) metabolism involving mitochondrial methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and its downstream purine synthesis pathway. MTHFD2 knockdown greatly reduced tumorigenesis and stem-like properties, which were associated with purine nucleotide deficiency, and caused marked accumulation of 5-aminoimidazole carboxamide ribonucleotide (AICAR)-the final intermediate of the purine synthesis pathway. Lung cancer cells with acquired resistance to the targeted drug gefitinib, caused by elevated expression of components of the β-catenin pathway, exhibited increased stem-like properties and enhanced expression of MTHFD2. MTHFD2 knockdown or treatment with AICAR reduced the stem-like properties and restored gefitinib sensitivity in these gefitinib-resistant cancer cells. Moreover, overexpression of MTHFD2 in gefitinibsensitive lung cancer cells conferred resistance to gefitinib. Thus, MTHFD2-mediated mitochondrial 1C metabolism appears critical for cancer stem-like properties and resistance to drugs including gefitinib through consumption of AICAR, leading to depletion of the intracellular pool of AICAR. Because CSCs are dependent on MTHFD2, therapies targeting MTHFD2 may eradicate tumors and prevent recurrence.
Cancer stem cells are thought to be responsible for tumor growth, recurrence, and resistance to c... more Cancer stem cells are thought to be responsible for tumor growth, recurrence, and resistance to conventional cancer therapy. However, it is still unclear how they are maintained in tumor tissues. Here, we show that the growth differentiation factor 15 (GDF15), a member of the TGFβ family, may maintain cancer stem-like cells in breast cancer tissues by inducing its own expression in an autocrine/paracrine manner. We found that GDF15, but not TGFβ, increased tumor sphere formation in several breast cancer cell lines and patient-derived primary breast cancer cells. As expected, TGFβ strongly stimulated the phosphorylation of Smad2. GDF15 also stimulated the phosphorylation of Smad2, but the GDF15-induced tumor sphere forming efficiency was not significantly affected by treatment with SB431542, an inhibitor of the TGFβ signaling. Although TGFβ transiently activated ERK1/2, GDF15 induced prolonged activation of ERK1/2. Treatment with U0126, an inhibitor of the MEK-ERK1/2 signaling, great...
Tumor cells respond to their microenvironment, which can include hypoxia and malnutrition, and ad... more Tumor cells respond to their microenvironment, which can include hypoxia and malnutrition, and adapt their metabolism to survive and grow. Some oncogenes are associated with cancer metabolism via regulation of the related enzymes or transporters. However, the importance of metabolism and precise metabolic effects of oncogenes in colorectal cancer remain unclear. We found that colorectal cancer cells survived under the condition of glucose depletion, and their resistance to such conditions depended on genomic alterations rather than on KRAS mutation alone. Metabolomic analysis demonstrated that those cells maintained tricarboxylic acid cycle activity and ATP production under such conditions. Furthermore, we identified pivotal roles of GLUD1 and SLC25A13 in nutritional stress. GLUD1 and SLC25A13 were associated with tumor aggressiveness and poorer prognosis of colorectal cancer. In conclusion, GLUD1 and SLC25A13 may serve as new targets in treating refractory colorectal cancer which s...
In vivo studies: Sprague-Dawley rats, 300-400 g, were anaesthetised with α-chloralose and ketamin... more In vivo studies: Sprague-Dawley rats, 300-400 g, were anaesthetised with α-chloralose and ketamine and colonic intraluminal pressure and blood pressure were recorded. In other experiments, visceromotor responses to colorectal distension (VMR) were measured by recording the EMG from abdominal muscle. Fecal output was measured from conscious rats housed in a quiet environment. Immunohistochemistry was done on spinal cord sections and receptor and peptide gene expression were determined by RT-PCR. Results Receptor immunoreactivity occurred in the cell bodies of autonomic pre-ganglionic neurons at all levels of the spinal cord, from T1 to S2. RT-PCR from all levels of the cord detected transcripts for both the ghrelin receptor and ghrelin itself. Intravenous infusion of CP-464709 (5-20 mg/kg/h) caused propulsive contractions of the colon that was associated with increased propulsion of colonic contents in anaesthetised rats, as well as a blood pressure rise. The contractions induced by the agonist were large and regular, but it had no direct effect on the muscle. In conscious rats, subcutaneous CP464709 caused fecal expulsion. Intrathecal application of CP464709 or ghrelin at L6-S1, the region of defecation control centres, but not application at ponto-medullary levels, elicited propulsive contractions. The agonists administered to the thoracic cord raised blood pressure by 40 mm Hg. The colonic motility evoked by intravenous CP464709 was prevented if the pelvic nerve outflows were severed. When high dose of CP464709, applied intrathecally, was used to desensitise its receptors, the effect of intravenous CP464709 was blocked. CP464709 did not affect small intestine motility or the amplitudes of visceromotor reflexes caused by colorectal distension. Conclusions Activation of ghrelin receptors in the lumbo-sacral spinal cord triggers coordinated propulsive contractions that empty the colon. The pathways through which these responses are generated pass out of the spinal cord via the pelvic nerves and cause propulsive contractions through activation of enteric neurons. Activation of ghrelin receptors in thoracic and lumbo-sacral regions increases blood pressure.
From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently ... more From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3 clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth facto...
The fibroblast growth factor receptor substrate (FRS)-2 protein family comprises FRS2a, a wellkno... more The fibroblast growth factor receptor substrate (FRS)-2 protein family comprises FRS2a, a wellknown central mediator for fibroblast growth factor signaling, and FRS2b, whose endogenous expression pattern and function are not yet defined. Immunohistochemical analysis revealed that expression of FRS2b was restricted to neural tissues and it colocalized with Tuj1, a neuronal marker. There are two distinct patterns of FRS2b expression in neural cells: punctate and cup/ring-shaped; moreover, some particles colocalized with lysosomes. Stimulation with brain-derived neurotrophic factor (BDNF) enhanced FRS2b phosphorylation and the cup/ring-shaped pattern. These results suggest a probable role of FRS2b in the intracellular degradation systems of neural cells, which involves lysosomes.
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 1994
Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a liga... more Epidermal growth factor (EGF) receptor (EGFR) can induce cell growth and transformation in a ligand-dependent manner. To examine whether the autophosphorylation of EGFR correlates with the capacity of the
The CD74-Neuregulin1 (NRG1) fusion gene was recently identified as novel driver of invasive mucin... more The CD74-Neuregulin1 (NRG1) fusion gene was recently identified as novel driver of invasive mucinous adenocarcinoma, a malignant form of lung cancer. However, the function of the CD74-NRG1 fusion gene in adenocarcinoma pathogenesis and the mechanisms by which it may impart protumorigenic characteristics to cancer stem cells (CSC) is still unclear. In this study, we found that the expression of the CD74-NRG1 fusion gene increased the population of lung cancer cells with CSC-like properties. CD74-NRG1 expression facilitated sphere formation not only of cancer cells, but also of nonmalignant lung epithelial cells. Using a limiting dilution assay in a xenograft model, we further show that the CD74-NRG1 fusion gene enhanced tumor initiation. Mechanistically, we found that CD74-NRG1 expression promoted the phosphorylation of ErbB2/3 and activated the PI3K/Akt/NF-κB signaling pathway. Furthermore, the expression of the secreted insulin-like growth factor 2 (IGF2) and phosphorylation of its...
Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer and is the most common and f... more Non-small cell lung cancer (NSCLC) is a major subtype of lung cancer and is the most common and fatal cancer worldwide. Specific tyrosine kinase inhibitors for epidermal growth factor receptor (EGFR), such as gefitinib, have been effective in some NSCLC patients and are being used in the clinical setting as pioneer molecularly targeted cancer drugs. However, many patients have not responded to these drugs, and have acquired resistance after long-term treatment. To identify other potential NSCLC molecular targets, we used DNA microarrays to examine gene expression profiles of gefitinib-resistant PC9/ZD cells that are derived from gefitinib-sensitive PC9 cells and harbor a threonine to methionine mutation at codon 790 (T790M) in EGFR, a known mechanism of acquired resistance to gefitinib. We found that N-cadherin expression was significantly upregulated in PC9/ZD cells compared with PC9 cells. Inhibition of N-cadherin expression by siRNA or treatment with antibodies against N-cadherin induced apoptosis of PC9/ZD cells in association with reduced phosphorylation of Akt and Bad, a proapoptotic protein. Moreover, inhibition of Akt expression by siRNA or treatment with an inhibitor for phosphatidylinositol (PI)-3 kinase reduced survival of PC9/ZD cells. In addition, we found several N-cadherin-expressing lung cancer cells that showed inherent resistance to gefitinib treatment and reduced survival owing to siRNA-induced inhibition of N-cadherin expression. Thus, it appears that N-cadherin maintains the survival of the gefitinib-resistant lung cancer cells via the PI-3 kinase/Akt survival pathway. From these results, we propose that N-cadherin signaling contributes, at least in part, to the survival mechanisms of gefitinib-resistant NSCLC cells and that N-cadherin is a potential molecular target in the treatment of NSCLC.
Interleukin 3 (IL-3) not only induces DNA synthesis of haematopoietic cells but also maintains th... more Interleukin 3 (IL-3) not only induces DNA synthesis of haematopoietic cells but also maintains their viability by suppressing apoptosis. IL-3 stimulates tyrosine phosphorylation of the Shc adaptor protein and thereby formation of a complex of Shc with Grb2 at phosphorylated tyrosine (Y) residue 317-Shc. This pathway is implicated in Ras/mitogen-activated protein kinase (MAPK) activation towards c-fos gene expression. We examined the possible involvement of Shc in the antiapoptotic activity of IL-3. Conditional overexpression of the Shc SH2 domain, a dominant-negative mutant of Shc, was found to induce apoptosis of IL-3-dependent BaIF3 cells along with a reduction of c-myc gene expression. Apoptosis was rescued by the exogenously introduced c-myc gene. Since we identify novel tyrosine phosphorylation sites of Shc: Y239 and Y240, their role on cell survival was tested by mutational analysis. Ba/F3 cells expressing mutant Shc Y317F, which is unable to stimulate efficiently the Ras pathway, still showed resistance to apoptosis. However, cells expressing Shc Y239/240F, which is able to stimulate the Ras pathway, were sensitive to apoptosis. In these cells, induction of the c-myc gene was reduced. These findings suggest that a new signalling pathway for cell survival is generated from Y239/240 of Shc to the nuclei involving c-myc gene expression.
Unlike other cyclins that positively regulate the cell cycle, cyclin G2 (CCNG2) regulates cell pr... more Unlike other cyclins that positively regulate the cell cycle, cyclin G2 (CCNG2) regulates cell proliferation as a tumor suppressor gene. A decreased CCNG2 expression serves as a marker for poor prognosis in several types of cancer. The aim of the present study was to clarify the correlation of CCNG2 expression with overall survival and histopathological factors in pancreatic cancer patients. This retrospective analysis included data from 36 consecutive patients who underwent complete surgical resection for pancreatic cancer and did not undergo any preoperative therapies. The association between prognoses and the expression of CCNG2 was assessed using immunohistochemical staining. Multivariate analysis identified that the expression of CCNG2 is an independent prognostic factor. In addition, the Kaplan-Meier curve for overall survival revealed that decreased expression of CCNG2 was a consistent indicator of poor prognosis in pancreatic cancer patients (P=0.0198). A decreased CCNG2 expression significantly correlated with venous invasion in tumor specimens and the tumor invasion depth. In conclusion, CCNG2 expression inversely reflected cancer progression and may be a novel, independent prognostic marker in pancreatic cancer.
CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Weste... more CRK belongs to a family of adaptor proteins that consist mostly of SH2 and SH3 domains. Far Western blotting with CRK SH3 has demonstrated that it binds to 135-to 145-, 160-, and 180-kDa proteins. The 135to 145-kDa protein is C3G, a CRK SH3-binding guanine nucleotide exchange protein. Here, we report on the molecular cloning of the 180-kDa protein, which is designated DOCK180 (180-kDa protein downstream of CRK). The isolated cDNA contains a 5,598-bp open reading frame encoding an 1,866-amino-acid protein. The deduced amino acid sequence did not reveal any significant homology to known proteins, except that an SH3 domain was identified at its amino terminus. To examine the function of DOCK180, a Ki-Ras farnesylation signal was fused to the carboxyl terminus of DOCK180, a strategy that has been employed successfully for activation of adaptor-binding proteins in vivo. Whereas wild-type DOCK180 accumulated diffusely in the cytoplasm and did not have any effect on cell morphology, farnesylated DOCK180 was localized on the cytoplasmic membrane and changed spindle 3T3 cells to flat, polygonal cells. These results suggest that DOCK180 is a new effector molecule which transduces signals from tyrosine kinases through the CRK adaptor protein.
Uploads
Papers by Noriko Gotoh