Amino Acid Substitution ; Antineoplastic Agents/pharmacology* ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics* ; Gastrointestinal Neoplasms/pathology* ; Humans ; MAP Kinase Signaling System/genetics* ; Mutation, Missense ; Receptor, ErbB-3/metabolism* ; Receptor, Fibroblast Growth Factor, Type 1/metabolism*

The locus coeruleus (LC) has been studied in major depressive disorder (MDD) and bipolar disorder (BD). A major problem of immunocytochemical studies in the human LC is interference with the staining of the immunocytochemical end-product by the omnipresent natural brown pigment neuromelanin. Here, we used a multispectral method to untangle the two colors: blue immunocytochemical staining and brown neuromelanin. We found significantly increased tyrosine hydroxylase (TH) in the LC of MDD patients-thus validating the method-but not in BD patients, and we did not find significant changes in the receptor tyrosine-protein kinase ErbB4 in the LC in MDD or BD patients. We observed clear co-localization of ErbB4, TH, and neuromelanin in the LC neurons. The different stress-related molecular changes in the LC may contribute to the different clinical symptoms in MDD and BD.
Aged ; Aged, 80 and over ; Bipolar Disorder ; metabolism ; pathology ; Depressive Disorder, Major ; metabolism ; pathology ; Female ; Humans ; Image Processing, Computer-Assisted ; Immunohistochemistry ; methods ; Locus Coeruleus ; metabolism ; pathology ; Male ; Melanins ; metabolism ; Microscopy ; methods ; Middle Aged ; Neurons ; metabolism ; pathology ; Receptor, ErbB-4 ; metabolism ; Sensitivity and Specificity ; Spectrum Analysis ; methods ; Tyrosine 3-Monooxygenase ; metabolism

BACKGROUNDThe addition of anti-human epidermal growth factor receptor 2 (HER2)-targeted drugs, such as trastuzumab, lapatinib, and trastuzumab emtansine (T-DM1), to chemotherapy significantly improved prognosis of HER2-positive breast cancer patients. However, it was confused that metastatic patients vary in the response of targeted drug. Therefore, methods of accurately predicting drug response were really needed. To overcome the spatial and temporal limitations of biopsies, we aimed to develop a more sensitive and less invasive method of detecting mutations associated with anti-HER2 therapeutic response through circulating-free DNA (cfDNA).

METHODSFrom March 6, 2014 to December 10, 2014, 24 plasma samples from 20 patients with HER2-positive metastatic breast cancer who received systemic therapy were eligible. We used a panel for detection of hot-spot mutations from 50 oncogenes and tumor suppressor genes, and then used targeted next-generation sequencing (NGS) to identify somatic mutation of these samples in those 50 genes. Samples taken before their first trastuzumab administration and subsequently proven with clinical benefit were grouped into sensitive group. The others were collected after disease progression of the trastuzumab-based therapy and were grouped into the resistant group.

RESULTSA total of 486 single-nucleotide variants from 46 genes were detected. Of these 46 genes, phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA), proto-oncogene c-Kit (KIT), and tumor protein p53 (TP53) were the most common mutated genes. Seven genes, including epidermal growth factor receptor (EGFR), G protein subunit alpha S (GNAS), HRas proto-oncogene (HRAS), mutL homolog 1 (MLH1), cadherin 1 (CDH1), neuroblastoma RAS viral oncogene homolog (NRAS), and NOTCH1, that only occurred m utations in the resistant group were associated with the resistance of targeted therapy. In addition, we detected a HER2 S855I mutation in two patients who had persistent benefits from anti-HER2 therapy.

CONCLUSIONTargeted NGS of cfDNA has potential clinical utility to detect biomarkers from HER2-targeted therapies.

Adolescent ; Adult ; Aged ; Biomarkers, Tumor ; genetics ; Breast Neoplasms ; genetics ; metabolism ; Cadherins ; genetics ; Chromogranins ; genetics ; Class I Phosphatidylinositol 3-Kinases ; Drug Resistance, Neoplasm ; genetics ; Female ; GTP-Binding Protein alpha Subunits, Gs ; genetics ; Humans ; Male ; Middle Aged ; Mutation ; genetics ; Phosphatidylinositol 3-Kinases ; genetics ; Proto-Oncogene Proteins c-kit ; genetics ; Receptor, ErbB-2 ; metabolism ; Receptor, Notch1 ; genetics ; Tumor Suppressor Protein p53 ; genetics ; Young Adult

PURPOSE: To examine the usefulness of various receptor tyrosine kinase expressions as prognostic markers and therapeutic targets in muscle invasive urothelial cancer (UC) patients. MATERIALS AND METHODS: We retrospectively analyzed the data of 98 patients with muscle invasive UC who underwent radical cystectomy between 2005 and 2010 in Yonsei Cancer Center. Using formalin fixed paraffin embedded tissues of primary tumors, immunohistochemical staining was done for human epidermal growth factor receptor 2 (HER2), fibroblast growth factor receptor 1 (FGFR1), and fibroblast growth factor receptor 3 (FGFR3). RESULTS: There were 41 (41.8%), 44 (44.9%), and 14 (14.2%) patients who have over-expressed HER2, FGFR1, and FGFR3, respectively. In univariate analysis, significantly shorter median time to recurrence (TTR) (12.9 months vs. 49.0 months; p=0.008) and overall survival (OS) (22.3 months vs. 52.7 months; p=0.006) was found in patients with FGFR1 overexpression. By contrast, there was no difference in TTR or OS according to the HER2 and FGFR3 expression status. FGFR1 remained as a significant prognostic factor for OS with hazard ratio of 2.23 (95% confidence interval: 1.27-3.90, p=0.006) in multivariate analysis. CONCLUSION: Our result showed that FGFR1 expression, but not FGFR3, is an adverse prognostic factor in muscle invasive UC patients after radical cystectomy. FGFR1 might be feasible for prognosis prediction and a potential therapeutic target after thorough validation in muscle invasive UC.
Adult ; Aged ; Aged, 80 and over ; Carcinoma/*metabolism/*mortality/surgery ; Cystectomy ; Female ; Humans ; Male ; Middle Aged ; Multivariate Analysis ; Muscles/pathology ; Neoplasm Invasiveness ; Prognosis ; Proportional Hazards Models ; Receptor, ErbB-2/metabolism ; Receptor, Fibroblast Growth Factor, Type 1/*metabolism ; Receptor, Fibroblast Growth Factor, Type 3/metabolism ; Retrospective Studies ; Survival Rate ; Urinary Bladder Neoplasms/*metabolism/*mortality/surgery ; Urothelium/pathology

OBJECTIVETo study the association of human epidermal growth factor receptor family molecules expression in gastric cancer tissues with the prognosis of patients with gastric cancer.

METHODSClinical data of 161 patients with gastric cancer undergoing gastrectomy in Jiangsu Cancer Hospital between January 2006 and January 2007 were analyzed retrospectively. The expression of HER1, HER2, HER3 and HER4 was detected by immunohistochemistry. Association of the expression of HER family with the prognosis of patients was examined. Kaplan-Meier method was used to analyze the survival.

RESULTSHigh expression rates of HER1, HER2, HER3 and HER4 were 46.0% (74/161), 10.6% (17/161),55.9% (90/161) and 68.3% (110/161) respectively. Univariate analysis revealed that high expression of HER3 was associated with tumor invasion depth, lymph node metastasis, stage, neurovascular invasion, and overall 4-year survival. High expression of HER4 was associated with tumor distant metastasis and stage. High co-expression of HER2 and HER3 was associated with overall 4-year survival (P=0.023). Multivariate analysis revealed that high expression of HER3 and stage were prognostic independent factors.

CONCLUSIONUp-regulated expression of HER3 is associated with the poor prognosis in gastric cancer patients.

Adult ; Aged ; Aged, 80 and over ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; Middle Aged ; Prognosis ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptor, ErbB-3 ; metabolism ; Receptor, ErbB-4 ; Retrospective Studies ; Stomach Neoplasms ; metabolism ; pathology

In the present study, we investigate the expression profile of the epidermal growth factor receptor family, which comprises EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4 in oral leukoplakia (LP). The expression of four epidermal growth factor receptor (EGFR) family genes and their ligands were measured in LP tissues from 14 patients and compared with levels in 10 patients with oral lichen planus (OLP) and normal oral mucosa (NOM) from 14 healthy donors by real-time polymerase chain reaction (PCR) and immunohistochemistry. Synchronous mRNA coexpression of ErbB1, ErbB2, ErbB3 and ErbB4 was detected in LP lesions. Out of the receptors, only ErbB4 mRNA and protein was more highly expressed in LP compared with NOM tissues. These were strongly expressed by epithelial keratinocytes in LP lesions, as shown by immunohistochemistry. Regarding the ligands, the mRNA of Neuregulin2 and 4 were more highly expressed in OLP compared with NOM tissues. Therefore, enhanced ErbB4 on the keratinocytes and synchronous modulation of EGFR family genes may contribute to the pathogenesis and carcinogenesis of LP.
Adult ; Aged ; Amphiregulin ; Betacellulin ; EGF Family of Proteins ; Epidermal Growth Factor ; metabolism ; Epiregulin ; Female ; Gene Expression Profiling ; Glycoproteins ; metabolism ; Heparin ; metabolism ; Heparin-binding EGF-like Growth Factor ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Keratinocytes ; metabolism ; Leukoplakia, Oral ; metabolism ; Lichen Planus, Oral ; metabolism ; Ligands ; Male ; Middle Aged ; Mouth Mucosa ; metabolism ; Nerve Growth Factors ; Neuregulins ; metabolism ; RNA, Messenger ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptor, ErbB-3 ; metabolism ; Receptor, ErbB-4 ; Receptors, Cell Surface ; metabolism ; Transforming Growth Factor alpha ; metabolism ; Up-Regulation ; physiology

OBJECTIVETo study the molecular risk factors of lymph node metastasis in stage T1 and T2 colorectal cancers by tissue microarray and immunohistochemistry techniques.

METHODSTwo hundred and three patients with stage T1 and T2 colorectal carcinoma who underwent radical surgery from 1999 to 2010 in our department were included in this study. Their clinicopathological data were retrospectively analyzed. Expression of the following 14 molecular markers were selected and assayed by tissue microarray and immunohistochemistry: VEGFR-3, HER2, CD44v6, CXCR4, TIMP-1, EGFR, IGF-1R, IGF-2, IGFBP-1, ECAD, MMP-9, RKIP, CD133, MSI. Chi-squared test and logistic regression were used to evaluate the variables as potential risk factors for lymph node metastasis.

RESULTSThe positive expression rates of biomarkers were as following: VEGFR-3 (44.3%), EGFR (30.5%), HER-2 (28.1%), IGF-1R (63.5%), IGF-2 (44.8%), IGFBP-1 (70.9%), ECAD (45.8%), CD44v6 (51.2%), MMP-9 (44.3%), TIMP-1 (41.4%), RKIP (45.3%), CXCR4 (40.9%), and CD133 (49.8%). The positive rate of MSI expression was 22.2%. Both univariate and multivariate analyses showed that VEGFR-3, HER-2, and TIMP-1 were significant predictors of lymph node metastasis. Univariate analysis showed that CD44v6 and CXCR4 were significant significant predictors of lymph node metastasis.

CONCLUSIONSVEGFR-3, HER2 and TIMP-1 are independent factors for lymph node metastasis in stage T1 and T2 colorectal cancers.

Aged ; Biomarkers, Tumor ; metabolism ; Colonic Neoplasms ; metabolism ; pathology ; Female ; Humans ; Hyaluronan Receptors ; metabolism ; Immunohistochemistry ; Lymphatic Metastasis ; Male ; Microsatellite Instability ; Middle Aged ; Neoplasm Staging ; Oligonucleotide Array Sequence Analysis ; Receptor, ErbB-2 ; metabolism ; Receptors, CXCR4 ; metabolism ; Rectal Neoplasms ; metabolism ; pathology ; Retrospective Studies ; Tissue Inhibitor of Metalloproteinase-1 ; metabolism ; Vascular Endothelial Growth Factor Receptor-3 ; metabolism

OBJECTIVETo investigate the effect of gefitinib on the migration of triple-negative breast cancer cell line MDA-MB-231 cells.

METHODSGefitinib was used in concentrations of 0 micromol/L, 0.1 micromol/L, 1 micromol/L, 10 micromol/L and 20 micromol/L, respectively. Phosphorylation levels of EGFR and Akt were analyzed by Western blot. The capability of migration was measured by scratch test and Boyden chamber assay. Microfilaments (cell skeleton ) remolding and polarization were evaluated by immunofluorescence microscopy.

RESULTSComparing with the control group (0 micromol/L gefitinib), gefitinib effectively inhibited the phosphorylation of EGFR and its downstream key proteins, and the effect displayed an obvious dose-effect relationship. At 24 hours after wound scratch, the cell migration distance of each group with 0, 0.1, 1, 10, 20 micromol/L gefitinib was (36.3 +/- 4.0) microm, (30.3 +/- 3.8) microm, (26.8 +/- 3.3) microm, (17.0 +/- 2.6) microm, and (11.0 +/- 2.5) microm, respectively. At 3.5 hours after Boyden chamber assay, the cell count of each group with 0, 0.1, 1, 10, 20 micromol/L gefitinib was 69.2 +/- 7.0, 51.8 +/- 7.5, 43.8 +/- 8.7, 30.6 +/- 4.8, and 28.4 +/- 3.4, respectively. Compared with the control group (0 micromol/L gefitinib), gefitinib could significantly prolong the wound-healing time and decrease the migrating cell count (P < 0.05), and significantly inhibit the lamellipodium formation, cell skeleton remolding and changes of the cytoskeleton polarization.

CONCLUSIONSGefitinib can reduce the migration capacity of triple-negative breast cancer cells through inhibiting phosphorylation of EGFR/PI3K/Akt pathway, suppressing the cell skeleton (microfilaments) remolding and changes of its polarization.

Antineoplastic Agents ; administration & dosage ; pharmacology ; Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cytoskeleton ; drug effects ; Dose-Response Relationship, Drug ; Female ; Humans ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Protein Kinase Inhibitors ; administration & dosage ; pharmacology ; Proto-Oncogene Proteins c-akt ; metabolism ; Quinazolines ; administration & dosage ; pharmacology ; Receptor, Epidermal Growth Factor ; metabolism ; Receptor, ErbB-2 ; metabolism ; Receptors, Estrogen ; metabolism ; Receptors, Progesterone ; metabolism ; Signal Transduction ; drug effects

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2high/HER3 and the HER2low/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin-beta1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral-MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.
Breast Neoplasms/enzymology/*genetics/*metabolism ; Butadienes/pharmacology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Enzyme Inhibitors/pharmacology ; Female ; Gene Expression ; Gene Expression Regulation, Neoplastic/drug effects ; Humans ; MAP Kinase Signaling System ; MCF-7 Cells ; Matrix Metalloproteinase 1/*genetics/metabolism ; Matrix Metalloproteinase 9/*genetics/metabolism ; Neuregulin-1/*pharmacology ; Nitriles/pharmacology ; Phosphatidylinositol 3-Kinases/metabolism ; Protein Kinase Inhibitors/pharmacology ; Protein Multimerization ; Proto-Oncogene Proteins c-akt/metabolism ; Quinazolines/pharmacology ; Receptor, erbB-2/genetics/*metabolism ; Receptor, erbB-3/*metabolism

Dimerization among the EGFR family of tyrosine kinase receptors leads to allosteric activation of the kinase domains of the partners. Unlike other members in the family, the kinase domain of HER3 lacks key amino acid residues for catalytic activity. As a result, HER3 is suggested to serve as an allosteric activator of other EGFR family members which include EGFR, HER2 and HER4. To study the role of intracellular domains in HER3 dimerization and activation of downstream signaling pathways, we constructed HER3/HER2 chimeric receptors by replacing the HER3 kinase domain (HER3-2-3) or both the kinase domain and the C-terminal tail (HER3-2-2) with the HER2 counterparts and expressed the chimeric receptors in Chinese hamster ovary (CHO) cells. While over expression of the intact human HER3 transformed CHO cells with oncogenic properties such as AKT/ERK activation and increased proliferation and migration, CHO cells expressing the HER3-2-3 chimeric receptor showed significantly reduced HER3/HER2 dimerization and decreased phosphorylation of both AKT and ERK1/2 in the presence of neuregulin-1 (NRG-1). In contrast, CHO cells expressing the HER3-2-2 chimeric receptor resulted in a total loss of downstream AKT activation in response to NRG-1, but maintained partial activation of ERK1/2. The results demonstrate that the intracellular domains play a crucial role in HER3's function as an allosteric activator and its role in downstream signaling.
Amino Acid Sequence ; Animals ; CHO Cells ; Cell Movement ; Cell Proliferation ; Cricetinae ; Cricetulus ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Humans ; Intracellular Space ; enzymology ; MAP Kinase Signaling System ; Models, Molecular ; Molecular Sequence Data ; Phosphatidylinositol 3-Kinases ; metabolism ; Protein Multimerization ; Protein Structure, Quaternary ; Protein Structure, Tertiary ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptor, ErbB-2 ; chemistry ; Receptor, ErbB-3 ; chemistry ; genetics ; metabolism ; Recombinant Fusion Proteins ; chemistry ; genetics ; metabolism ; Signal Transduction