Patient-Derived Organoid Serves as a Platform for Personalized Chemotherapy in Advanced Colorectal Cancer Patients.

Background: Addition of oxaliplatin to adjuvant 5-FU has significantly improved the disease-free survival and served as the first line adjuvant chemotherapy in advanced colorectal cancer (CRC) patients. However, a fraction of patients remains refractory to oxaliplatin-based treatment. It is urgent to establish a preclinical platform to predict the responsiveness toward oxaliplatin in CRC patients as well as to improve the efficacy in the resistant patients. Methods: A living biobank of organoid lines were established from advanced CRC patients. Oxaliplatin sensitivity was assessed in patient-derived tumor organoids (PDOs) in vitro and in PDO-xenografted tumors in mice. Based on in vitro oxaliplatin IC50 values, PDOs were classified into either oxaliplatin-resistant (OR) or oxaliplatin-sensitive (OS) PDOs. The outcomes of patients undergone oxaliplatin-based treatment was followed. RNA-sequencing and bioinformatics tools were performed for molecular profiling of OR and OS PDOs. Oxaliplatin response signatures were submitted to Connectivity Map algorithm to identify perturbagens that may antagonize oxaliplatin resistance. Results: Oxaliplatin sensitivity in PDOs was shown to correlate to oxaliplatin-mediated inhibition on PDO xenograft tumors in mice, and parallelled clinical outcomes of CRC patients who received FOLFOX treatment. Molecular profiling of transcriptomes revealed oxaliplatin-resistant and -sensitive PDOs as two separate entities, each being characterized with distinct hallmarks and gene sets. Using Leave-One-Out Cross Validation algorithm and Logistic Regression model, 18 gene signatures were identified as predictive biomarkers for oxaliplatin response. Candidate drugs identified by oxaliplatin response signature-based strategies, including inhibitors targeting c-ABL and Notch pathway, DNA/RNA synthesis inhibitors, and HDAC inhibitors, were demonstrated to potently and effectively increase oxaliplatin sensitivity in the resistant PDOs. Conclusions: PDOs are useful in informing decision-making on oxaliplatin-based chemotherapy and in designing personalized chemotherapy in CRC patients.

Proteoglycan serglycin promotes non-small cell lung cancer cell migration through the interaction of its glycosaminoglycans with CD44.

BACKGROUND: Serglycin (SRGN), previously recognized as an intracellular proteoglycan involved in the storage processes of secretory granules, has recently been shown to be upregulated in several solid tumors. We have previously shown that SRGN in non-small cell lung cancer (NSCLC) promotes malignant phenotypes in a CD44-dependent manner and increased expression of SRGN predicts poor prognosis of primary lung adenocarcinomas. However, the underlying mechanism remains to be defined. METHODS: Overexpression, knockdown and knockout approaches were performed to assess the role of SRGN in cell motility using wound healing and Boyden chamber migration assays. SRGN devoid of glycosaminoglycan (GAG) modification was produced by site-directed mutagenesis or chondroitinase treatment. Liquid chromatography/tandem mass spectrometry was applied for quantitative analysis of the disaccharide compositions and sulfation extent of SRGN GAGs. Western blot and co-immunoprecipitation analyses were performed to determine the expression and interaction of proteins of interest. Actin cytoskeleton organization was monitored by immunofluorescence staining. RESULTS: SRGN expressed by NSCLC cells is readily secreted to the extracellular matrix in a heavily glycosylated form attached with mainly chondroitin sulfate (CS)-GAG chains, and to a lesser extent with heparin sulfate (HS). The CS-GAG moiety serves as the structural motif for SRGN binding to tumor cell surface CD44 and promotes cell migration. SRGN devoid of CS-GAG modification fails to interact with CD44 and has lost the ability to promote cell migration. SRGN/CD44 interaction promotes focal adhesion turnover via Src-mediated paxillin phosphorylation and disassembly of paxillin/FAK adhesion complex, facilitating cell migration. In support, depletion of Src activity or removal of CS-GAGs efficiently blocks SRGN-mediated Src activation and cell migration. SRGN also promotes cell migration via inducing cytoskeleton reorganization mediated through RAC1 and CDC42 activation accompanied with increased lamellipodia and filopodia formation. CONCLUSIONS: Proteoglycan SRGN promotes NSCLC cell migration via the binding of its GAG motif to CD44. SRGN/CD44 interaction induces Rho-family GTPase-mediated cytoskeleton reorganization and facilitates Src-mediated focal adhesion turnover, leading to increased cell migration. These findings suggest that targeting specific glycans in tumor microenvironment that serve as ligands for oncogenic pathways may be a potential strategy for cancer therapy.

ITPKA Gene Body Methylation Regulates Gene Expression and Serves as an Early Diagnostic Marker in Lung and Other Cancers.

INTRODUCTION: Despite recent advances in cancer therapy, the overall 5-year survival rate of patients with lung cancer remains low. The aim of our study was to search for novel markers for early diagnosis in patients with lung cancer. METHODS: Complementary DNA microarray analysis was performed in primary lung adenocarcinomas and cell lines to search for differentially expressed genes, followed by in vivo and in vitro tumorigenic assays to characterize the oncogenic potential of the candidate genes. Gene body methylation was analyzed by 450K methylation array, bisulfite sequencing, and quantitative methylation-specific polymerase chain reaction assays. In silico analysis of The Cancer Genome Atlas data set was also performed. RESULTS: Inositol-trisphosphate 3-kinase A gene (ITPKA), a kinase with limited tissue distribution, was identified as a potential oncogene. We showed that ITPKA expression is up-regulated in many forms of cancers, including lung and breast cancers, and that overexpressed ITPKA contributes to tumorigenesis. We also demonstrated that ITPKA expression is regulated by epigenetic DNA methylation of ITPKA gene body through modulation of the binding of SP1 transcription factor to the ITPKA promoter. ITPKA gene body displayed low or absent levels of methylation in most normal tissue but was significantly methylated in malignant tumors. In lung cancer, ITPKA gene body methylation first appeared at the in situ carcinoma stage and progressively increased after invasion. CONCLUSIONS: ITPKA is a potential oncogene that it is overexpressed in most tumors, and its overexpression promotes tumorigenesis. ITPKA gene body methylation regulates its expression and thus serves as a novel and potential biomarker for early cancer detection.

Functional assessment of glioma pathogenesis by in vivo multi-parametric magnetic resonance imaging and in vitro analyses.

Gliomas are aggressive brain tumors with poor prognosis. In this study, we report a novel approach combining both in vivo multi-parametric MRI and in vitro cell culture assessments to evaluate the pathogenic development of gliomas. Osteopontin (OPN), a pleiotropic factor, has been implicated in the formation and progression of various human cancers, including gliomas, through its functions in regulating cell proliferation, survival, angiogenesis, and migration. Using rat C6 glioma model, the combined approach successfully monitors the acquisition and decrease of cancer hallmarks. We show that knockdown of the expression of OPN reduces C6 cell proliferation, survival, viability and clonogenicity in vitro, and reduces tumor burden and prolongs animal survival in syngeneic rats. OPN depletion is associated with reduced tumor growth, decreased angiogenesis, and an increase of tumor-associated metabolites, as revealed by T2-weighted images, diffusion-weighted images, K(trans) maps, and 1H-MRS, respectively. These strategies allow us to define an important role of OPN in conferring cancer hallmarks, which can be further applied to assess the functional roles of other candidate genes in glioma. In particular, the non-invasive multi-parametric MRI measurement of cancer hallmarks related to proliferation, angiogenesis and altered metabolism may serve as a useful tool for diagnosis and for patient management.

Loss of PTPRM associates with the pathogenic development of colorectal adenoma-carcinoma sequence.

Identification and functional analysis of genes from genetically altered chromosomal regions would suggest new molecular targets for cancer diagnosis and treatment. Here we performed a genome-wide analysis of chromosomal copy number alterations (CNAs) in matching sets of colon mucosa-adenoma-carcinoma samples using high-throughput oligonucleotide microarray analysis. In silico analysis of NCBI GEO and TCGA datasets allowed us to uncover the significantly altered genes (p ≤ 0.001) associated with the identified CNAs. We performed quantitative PCR analysis of the genomic and complementary DNA derived from primary mucosa, adenoma, and carcinoma samples, and confirmed the recurrent loss and down-regulation of PTPRM in colon adenomas and carcinomas. Functional characterization demonstrated that PTPRM negatively regulates cell growth and colony formation, whereas loss of PTPRM promotes oncogenic cell growth. We further showed that, in accordance to Knudson's two-hit hypothesis, inactivation of PTPRM in colon cancer was mainly attributed to loss of heterozygosity and promoter hypermethylation. Taken together, this study demonstrates a putative tumor suppressive role for PTPRM and that genetic and epigenetic alterations of PTPRM may contribute to early step of colorectal tumorigenesis.

White matter tracts for the trafficking of neural progenitor cells characterized by cellular MRI and immunohistology: the role of CXCL12/CXCR4 signaling.

White matter tracts are important for the trafficking of neural progenitor cells (NPCs) in both normal and pathological conditions, but the underlying mechanism is not clear. The directionality of white matter is advantageous for molecules or cells to distribute over a long distance, but this feature is unlikely solely responsible for efficient migration. The present study hypothesizes that the efficient migration of NPCs into white matter is under the influences of neurochemical attraction­CXCL12/CXCR4 signaling, a major mechanism underlying the targeted migration of NPCs. To test this view, the present study investigated the effects of CXCL12 administration into the corpus callosum (CC) on the migratory behavior of transplanted NPCs. A living animal tracking platform based on MRI and a magnetic cell labeling technique was employed. The NPCs were magnetically labeled and then transplanted at the right end of the CC. CXCL12 was infused continuously at the left end. Migration of NPCs was monitored repeatedly over a 7-day course using 3D gradient echo T2*-weighted imaging. It was found that, CXCL12 induced NPCs to migrate up to 1,881 µm from the graft whereas the spontaneous migration was mere 200 µm. CXCL12 induced migration that was nine times as efficient in the speed. The results indicate that the CXCL12/CXCR4 signaling may be a mechanism via which NPCs efficiently migrate along the white matter tracts. The study also presents a potential strategy for facilitating the targeted migration in NPC therapy for brain disorders.

CSNK1E/CTNNB1 are synthetic lethal to TP53 in colorectal cancer and are markers for prognosis.

Two genes are called synthetic lethal (SL) if their simultaneous mutations lead to cell death, but each individual mutation does not. Targeting SL partners of mutated cancer genes can kill cancer cells specifically, but leave normal cells intact. We present an integrated approach to uncovering SL pairs in colorectal cancer (CRC). Screening verified SL pairs using microarray gene expression data of cancerous and normal tissues, we first identified potential functionally relevant (simultaneously differentially expressed) gene pairs. From the top-ranked pairs, ~20 genes were chosen for immunohistochemistry (IHC) staining in 171 CRC patients. To find novel SL pairs, all 169 combined pairs from the individual IHC were synergistically correlated to five clinicopathological features, e.g. overall survival. Of the 11 predicted SL pairs, MSH2-POLB and CSNK1E-MYC were consistent with literature, and we validated the top two pairs, CSNK1E-TP53 and CTNNB1-TP53 using RNAi knockdown and small molecule inhibitors of CSNK1E in isogenic HCT-116 and RKO cells. Furthermore, synthetic lethality of CSNK1E and TP53 was verified in mouse model. Importantly, multivariate analysis revealed that CSNK1E-P53, CTNNB1-P53, MSH2-RB1, and BRCA1-WNT5A were independent prognosis markers from stage, with CSNK1E-P53 applicable to early-stage and the remaining three throughout all stages. Our findings suggest that CSNK1E is a promising target for TP53-mutant CRC patients which constitute ~40% to 50% of patients, while to date safety regarding inhibition of TP53 is controversial. Thus the integrated approach is useful in finding novel SL pairs for cancer therapeutics, and it is readily accessible and applicable to other cancers.

Functional phosphoproteomics of oncogenic KRAS signaling.

Identification of oncogene-mediated phosphorylation events is essential to understanding the molecular determinants responsible for cancer development and progression. Here, we identify KRAS-regulated phosphorylation events using label-free quantitation-based comparative phosphoproteomics analyses of immortalized human bronchial epithelial cells that express oncogenic KRAS as well as cells that do not. Further, we demonstrate integration of the identified phosphorylation events with the Pathway Interaction Database to infer KRAS-regulated pathways, which may have implications in KRAS-associated lung adenocarcinoma development. Taken together, our study provides an overview of the functional phosphoproteomics approach involving cell culture, preparation of whole cell lysates, trypsin digestion, phosphopeptide enrichment, mass spectrometry analyses, label-free quantitative analyses, and signaling pathway analyses to study KRAS-targeted events.

Label-free quantitative proteomics and N-glycoproteomics analysis of KRAS-activated human bronchial epithelial cells.

Mutational activation of KRAS promotes various malignancies, including lung adenocarcinoma. Knowledge of the molecular targets mediating the downstream effects of activated KRAS is limited. Here, we provide the KRAS target proteins and N-glycoproteins using human bronchial epithelial cells with and without the expression of activated KRAS (KRAS(V12)). Using an OFFGEL peptide fractionation and hydrazide method combined with subsequent LTQ-Orbitrap analysis, we identified 5713 proteins and 608 N-glycosites on 317 proteins in human bronchial epithelial cells. Label-free quantitation of 3058 proteins (≥2 peptides; coefficient of variation (CV) ≤ 20%) and 297 N-glycoproteins (CV ≤ 20%) revealed the differential regulation of 23 proteins and 14 N-glycoproteins caused by activated KRAS, including 84% novel ones. An informatics-assisted IPA-Biomarker® filter analysis prioritized some of the differentially regulated proteins (ALDH3A1, CA2, CTSD, DST, EPHA2, and VIM) and N-glycoproteins (ALCAM, ITGA3, and TIMP-1) as cancer biomarkers. Further, integrated in silico analysis of microarray repository data of lung adenocarcinoma clinical samples and cell lines containing KRAS mutations showed positive mRNA fold changes (p < 0.05) for 61% of the KRAS-regulated proteins, including biomarker proteins, CA2 and CTSD. The most significant discovery of the integrated validation is the down-regulation of FABP5 and PDCD4. A few validated proteins, including tumor suppressor PDCD4, were further confirmed as KRAS targets by shRNA-based knockdown experiments. Finally, the studies on KRAS-regulated N-glycoproteins revealed structural alterations in the core N-glycans of SEMA4B in KRAS-activated human bronchial epithelial cells and functional role of N-glycosylation of TIMP-1 in the regulation of lung adenocarcinoma A549 cell invasion. Together, our study represents the largest proteome and N-glycoproteome data sets for HBECs, which we used to identify several novel potential targets of activated KRAS that may provide insights into KRAS-induced adenocarcinoma and have implications for both lung cancer therapy and diagnosis.

MR phase imaging: sensitive and contrast-enhancing visualization in cellular imaging.

The successful translation of stem-cell therapies requires a detailed understanding of the fate of transplanted cells. Magnetic resonance imaging (MRI) has provided a noninvasive means of imaging cell dynamics in vivo by prelabeling cell with T(2) shortening iron oxide particles. However, this approach suffers from a gradual loss of sensitivity since active cell mitosis could decrease the cellular contrast agent (CA) concentration below detection level. In addition, the interpretation of images may be confounded by hypointensities induced by factors other than this CA susceptibility effect (CASE). We therefore examined the feasibility of exploiting the phase information in MRI to increase the sensitivity of cellular imaging and to differentiate the CASE from endogenous image hypointensity. Phase aliasing and the B(0) field inhomogeneity effect were removed by applying a reliable unwrapping algorithm and a high-pass filter, respectively, thus delineating phase variations originating from high spatial frequencies due to the CASE. We found that the filtered phase map detects labeled cells with high sensitivity and can readily differentiate the cell migration track from the white matter, both of which are hypointense in T(2)-weighted magnitude images. Furthermore, an approximate fivefold contrast-to-noise ratio enhancement can be achieved with an MRI phase map over conventional T(2)-weighted magnitude images.

Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas.

BACKGROUND: Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale. METHODOLOGY/PRINCIPAL FINDINGS: By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells. CONCLUSIONS/SIGNIFICANCE: This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.

A 20-year retrospective study of small-cell carcinomas in Taiwan.

BACKGROUND AND OBJECTIVES: Small-cell carcinomas (SCC) develop most commonly in the lung (small-cell lung carcinoma, SCLC) and only small percentages are present at extra-pulmonary sites. This study aimed to examine the distribution, treatment, and survival of SCCs. METHODS: The records for 922 SCC cases of various origins between January 1989 and December 2008 were retrieved and analyzed. RESULTS: The lung (89.2%) was the most common location, followed by the esophagus (1.8%), urinary bladder (1.6%), uterine cervix (1.5%), colorectum (1.4%), skin (1.0%), stomach (0.9%), head and neck (0.7%), prostate (0.3%), and small intestine (0.1%). Limited disease (LD) SCLC patients underwent surgery and chemotherapy had significantly higher survival rates than those who received chemotherapy alone, those who underwent combined radiotherapy and chemotherapy, and those who were administered supportive treatment. Actuarial 1-, 2-, and 3-year survival rate was 28.9%, 9.4%, and 4.8% for total SCLC cases, 41.3%, 17.5%, and 9.6% for LD-SCLC patients, and 21.9%, 4.2%, and 1.8% for extensive disease (ED)-SCLC patients (P < 0.001). The survival rates for lung and stomach SCC patients with LD were significantly better than for patients with ED; cervical SCC stages I and IIa patients had better survival rates than patients with stage IIb and above (P = 0.034). CONCLUSION: The lung was the most common location of SCCs, with 9.3% of cases being extra-pulmonary in origin. The need for combined surgery and chemotherapy in LD-SCLC patients deserves further evaluation.

Small cell carcinomas in gastrointestinal tract: immunohistochemical and clinicopathological features.

AIMS: To test the incidence of the expression of the immunohistochemical markers that aid diagnosis of gastrointestinal tract small cell carcinoma (GI-SmCC) and to evaluate the incidence of mixed endocrine-exocrine carcinomas in GI-SmCC. METHODS: Immunohistochemical studies of three antibodies against epithelial markers (CK8, AE1/AE3, EMA), four neuroendocrine differentiation markers (synaptophysin (Syn), neuron specific enolase (NSE), neuronal cell adhesion molecules (CD56), chromogranin A (CgA)), and a transcription factor (thyroid transcription factor 1 (TTF-1)) were performed. The incidence of non-endocrine carcinoma component was evaluated in 42 GI-SmCCs (11 in the oesophagus, 15 in the stomach, 15 in the colon, and 1 in the small intestine). RESULTS: The percentages of GI-SmCC with positive immunoreactivity were: CK8 92.9%, AE1/AE3 76.2%, EMA 71.4%, Syn 100%, NSE 100%, CD56 90.5%, CgA 61.9%, TTF-1 21.4%. The low molecular weight cytokeratin CK8 is more commonly expressed in GI-SmCC than is the expression of AE1/AE3 or EMA. Synaptophysin and NSE are expressed in all GI-SmCCs studied. Non-endocrine carcinoma components were demonstrated in 8 patients (4 in the oesophagus and 4 in the stomach). CONCLUSION: In detecting GI-SmCC, epithelial marker CK8 is more sensitive than AE1/AE3 or EMA, and neuroendocrine differentiation markers synaptophysin and NSE are the most useful markers. TTF-1 positivity is not uncommon in GI-SmCC, but cases with negative TTF-1 staining may indicate an extra-pulmonary primary. Non-endocrine carcinoma components were demonstrated in about 30% of oesophagus and stomach SmCC; the neoplasms should be diagnosed as mixed endocrine-exocrine carcinoma.

IDEAL-Q, an automated tool for label-free quantitation analysis using an efficient peptide alignment approach and spectral data validation.

In this study, we present a fully automated tool, called IDEAL-Q, for label-free quantitation analysis. It accepts raw data in the standard mzXML format as well as search results from major search engines, including Mascot, SEQUEST, and X!Tandem, as input data. To quantify as many identified peptides as possible, IDEAL-Q uses an efficient algorithm to predict the elution time of a peptide unidentified in a specific LC-MS/MS run but identified in other runs. Then, the predicted elution time is used to detect peak clusters of the assigned peptide. Detected peptide peaks are processed by statistical and computational methods and further validated by signal-to-noise ratio, charge state, and isotopic distribution criteria (SCI validation) to filter out noisy data. The performance of IDEAL-Q has been evaluated by several experiments. First, a serially diluted protein mixed with Escherichia coli lysate showed a high correlation with expected ratios and demonstrated good linearity (R(2) = 0.996). Second, in a biological replicate experiment on the THP-1 cell lysate, IDEAL-Q quantified 87% (1,672 peptides) of all identified peptides, surpassing the 45.7% (909 peptides) achieved by the conventional identity-based approach, which only quantifies peptides identified in all LC-MS/MS runs. Manual validation on all 11,940 peptide ions in six replicate LC-MS/MS runs revealed that 97.8% of the peptide ions were correctly aligned, and 93.3% were correctly validated by SCI. Thus, the mean of the protein ratio, 1.00 +/- 0.05, demonstrates the high accuracy of IDEAL-Q without human intervention. Finally, IDEAL-Q was applied again to the biological replicate experiment but with an additional SDS-PAGE step to show its compatibility for label-free experiments with fractionation. For flexible workflow design, IDEAL-Q supports different fractionation strategies and various normalization schemes, including multiple spiked internal standards. User-friendly interfaces are provided to facilitate convenient inspection, validation, and modification of quantitation results. In summary, IDEAL-Q is an efficient, user-friendly, and robust quantitation tool. It is available for download.

SOCS6, down-regulated in gastric cancer, inhibits cell proliferation and colony formation.

Members of the suppressor of cytokine-induced signaling (SOCS) family are negative regulators of cytokine signaling pathways. By mRNA differential display, we showed that SOCS6 was frequently down-regulated in gastric cancer (GC). Our data showed that allelic loss and promoter hypermethylation may account for the major mechanisms leading to SOCS6 inactivation. Ectopic expression of SOCS6 suppressed cell growth and colony formation, in part through eliciting intrinsic apoptotic pathway, accompanied with decreased mitochondrial membrane potential. Taken together, this study provides molecular and functional data supporting the importance of loss-of-function of SOCS6 as a frequent event in gastric tumorigenesis.

Genomic organization and functional characterization of the promoter for the human suppressor of cytokine signaling 6 gene.

In this study, we report the expression and genomic structure of the gene encoding human suppressor of cytokine signaling 6 (SOCS6), and the characterization of the functional promoter region. The human SOCS6 gene, spanning 40 kb on chromosome 18q22.2, is composed of two exons separated by an intron of 35 kb. Two transcripts are ubiquitously expressed, and both encode the full-length open reading frame of SOCS6. A primer extension assay revealed that the major transcription initiation site is located 469 bp upstream the ATG codon. Luciferase promoter analysis demonstrated that the 5'-flanking region is able to drive transcription, and the CpG-rich sequences near the transcription initiation site are important for the TATA-less SOCS6 promoter activity. Analogous to SOCS1 and SOCS3, which are down-regulated in several human cancers, SOCS6 is expressed at lower levels in carcinomas of stomach and colon. We demonstrated that hypermethylation of the SOCS6 promoter is one of the mechanisms for the epigenetic regulation of SOCS6 expression. Firstly, in vitro methylation of the reporter promoter plasmid significantly suppressed the promoter activity. Secondly, SOCS6 expression in vivo was enhanced by treating cells with a methyltransferase inhibitor. The SOCS6 gene from various species shares significant homology in amino acid sequences, transcription factor binding motifs in promoter regions and the two-exon genomic structure, suggesting that the SOCS6 gene is highly conserved.

Acetylation and activation of STAT3 mediated by nuclear translocation of CD44.

Expression of the type I transmembrane glycoprotein CD44 has recently been recognized as a signature for cancer stem cells. In this study, we demonstrate that CD44, once engaged, is internalized and translocated to the nucleus, where it binds to various promoters, including that of cyclin D1, leading to cell fate change through transcriptional reprogramming. In regulating cyclin D1 expression, the internalized CD44 forms a complex with STAT3 and p300 (acetyltransferase), eliciting STAT3 acetylation at lysine 685 and dimer formation in a cytokine- and growth factor-independent manner. A bipartite nuclear localization signal (NLS) was mapped to the cytoplasmic tail of CD44, which mediates its nuclear translocation. Expression of CD44(NLS) mutant sequesters STAT3 in cytosol. In the nucleus, the acetylated STAT3 dimer remains associated with CD44 and binds to the cyclin D1 promoter, leading to increased cyclin D1 expression and cell proliferation. This study describes a novel function for CD44 in transcriptional modulation through nuclear translocation of the internalized CD44 and complex formation with transcription factors.

Alterations in the p16INK4a/cyclin D1/RB pathway in gastrointestinal tract endocrine tumors.

A series of 76 cases of gastrointestinal tract endocrine tumors, including 21 poorly differentiated endocrine carcinomas (PDECs, small cell carcinomas) and 55 well-differentiated endocrine neoplasms (WDENs, carcinoids), 18 metastatic and 37 nonmetastatic rectal carcinoids, were examined by immunohistochemical analysis for p16INK4a, cyclin D1, and retinoblastoma protein (pRB) expression. Overexpression of p16INK4a was noted in 16 (76%) of the PDECs and none of the WDENs (P < .0001). Loss of pRB expression was demonstrated in 14 (67%) of the PDECs and 17 (31%) of the WDENs (P = .004). Overexpression of cyclin D1 was noted in 49 (89%) of the WDENs and 3 (14%) of the PDECs (P < .0001). Loss of pRB expression was noted in 11 (61%) of 18 metastatic WDENs and only 6 (16%) of 37 nonmetastatic WDENs (P = .001). The p16INK4a/cyclin D1/pRB pathway was altered in gastrointestinal tract endocrine tumors, and the loss of expression of pRB may be helpful in identifying patients at high risk of metastasis in rectal WDENs.

Immobilized metal affinity chromatography revisited: pH/acid control toward high selectivity in phosphoproteomics.

Despite recent advances in instrumentation and analytical strategies for identification and quantitation of protein phosphorylation, a highly specific enrichment protocol is still a challenge in large-scale studies. Here, we report a simple pH/acid control method that addresses the poor specificity seriously criticized in IMAC. Detailed evaluation of the capture and release mechanism in IMAC revealed that pH, buffer and salt yield a complex interplay in enrichment of phosphopeptides, yet they play individual roles in recovery and specificity. A revised one-step IMAC method with low sample loss and high specificity can be rationally designed by controlling salt, pH and the structure and concentration of organic acid. Without methyl esterification, the one-step IMAC enrichment with single LC-MS/MS identified 386 phosphoproteins in 550 mug of non-small-cell lung cancer cell lysate with 96% specificity. Additional fractionation by SDS-PAGE from 4 mg of cell lysate revealed the comprehensive proteome map, identifying 2747 phosphorylation sites from 2360 nondegenerate phosphopeptides and 1219 phosphoproteins with a false discovery rate of 0.63%. To our knowledge, this pH/acid-controlled IMAC procedure provides higher specificity than any other one-step IMAC purification procedure. Furthermore, the simple and reproducible IMAC protocol can be adapted to other solid supports, fully automated or manual, for large-scale identification of the vastly under-explored phosphoproteome.

CD44 engagement promotes matrix-derived survival through the CD44-SRC-integrin axis in lipid rafts.

CD44 is present in detergent-resistant, cholesterol-rich microdomains, called lipid rafts, in many types of cells. However, the functional significance of CD44 in lipid rafts is still unknown. We have previously demonstrated that osteopontin-mediated engagement of CD44 spliced variant isoforms promotes an extracellular matrix-derived survival signal through integrin activation. By using a series of CD44 mutants and pharmacological inhibitors selectively targeted to various cellular pathways, we show in this study that engagement of CD44 induces lipid raft coalescence to facilitate a CD44-Src-integrin signaling axis in lipid rafts, leading to increased matrix-derived survival. Palmitoylation of the membrane-proximal cysteine residues and carboxyl-terminal linkage to the actin cytoskeleton both contribute to raft targeting of CD44. The enrichment of integrin beta1 in lipid rafts is tightly coupled to CD44 ligation-elicited lipid raft reorganization and associated with temporally delayed endocytosis. Through the interaction with the CD44 carboxyl-terminal ankyrin domain, Src is cotranslocated to lipid rafts, where it induces integrin activation via an inside-out mechanism. Collectively, this study demonstrates an important role of the dynamic raft reorganization induced by CD44 clustering in eliciting the matrix-derived survival signal.