Nuclear-cytoplasmic translocation of SQSTM1/p62 protein enhances ESCC cell migration and invasion by stabilizing EPLIN expression.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignant disease with a poor prognosis. We previously found that p62 presented a marked nuclear-cytoplasmic translocation in ESCC cells as compared that in normal esophageal epithelial cells, but its effects on ESCC cells remain unclear. This study aims to clarify the impacts of different cellular localization of p62 on the function of ESCC cells and the underlying molecular mechanisms. We here demonstrated that cytoplasmic p62 enhances the migration and invasion abilities of esophageal cancer cells, whereas nuclear p62 has no effect. We further explored the interaction protein of p62 by using GST pull-down experiment and identified EPLIN as a potential protein interacting with p62. In addition, reducing EPLIN expression significantly inhibited the migration and invasion of ESCC cells, which were rescued when EPLIN expression was restored after the p62 knockdown. At a molecular level, p62 in cytoplasm positively regulated the expression of EPLIN via enhancing its protein stability. Data from the TCGA and GEO database displayed a significant up-regulation of EPLIN mRNA expression in ESCC tissues compared with corresponding paired esophageal epithelial samples. Our findings present evidence that the nuclear-cytoplasmic translocation of p62 protein contributes to an aggressive malignancy phenotype, providing candidate molecular biomarkers and potential molecular targets for the diagnosis and treatment of ESCC.

Dysregulation of CXCL14 promotes malignant phenotypes of esophageal squamous carcinoma cells via regulating SRC and EGFR signaling.

The present study was to identify abnormal methylation genes implicated in esophageal squamous cell carcinoma (ESCC). Genomic methylation alterations in ESCC tissues were analyzed using laser-microdissection and whole-genome bisulfite sequencing. CXCL14 promoter was frequently hypermethylated in ESCC tissues. The correlation of CXCL14 hypermethylation status and the mRNA and protein expression levels were validated using nested methylation-specific PCR (nMS-PCR), RNAscope in situ hybridization (RISH) and Western blot. RISH results showed completely negative CXCL14 expression in 34.3% (34/99) ESCC, compared with those in the basal layer cells of normal epithelia. Low expression of CXCL14 was more present in patients with lower differentiation. The anticancer role of CXCL14 has been commonly associated with immune regulation in the literature. Here, we observed by functional analysis that CXCL14 can also act as a tumor suppressor in ESCC cells. 5-Aza-dC treatment suppressed CXCL14 methylation and up-regulated the expression of CXCL14. Ectopic expression of CXCL14 suppressed the proliferation, invasion, tumor growth, and lung metastasis of ESCC cells. Both ectopic expression and induction of CXCL14 with 5-Aza-dC inhibited the activity of SRC, MEK1/2 and STAT3 in ESCC cells, while activated EGFR. Importantly, a combination of CXCL14 expression and SRC or EGFR inhibitor dramatically repressed the proliferation of ESCC cells and the growth of xenografts. Our findings revealed a direct tumor suppressor role of CXCL14, but not through the immune system. The data suggest that for ESCC patients with low level CXCL14, increasing CXCL14 expression combined with inhibition of SRC or EGFR might be a promising therapeutic strategy.

NEFL promotes invasion and migration of esophageal squamous carcinoma cells via the EGFR/AKT/S6 pathway.

To explore the expression, the roles and the underlying mechanism of neurofilament light chain (NEFL) in esophageal squamous cell carcinoma (ESCC), we firstly analyzed the NEFL mRNA and protein expression in ESCC and paired normal tissues by using Gene Expression Omnibus (GEO) database, and real-time quantitative reverse transcription PCR (qRT-PCR). The results showed that NEFL mRNA level was significantly upregulated in ESCC tissues compared with that of normal tissues. Western blot analysis revealed that NEFL protein level was also significantly upregulated in ESCC tissues. CCK8 and transwell assays were performed to analyze the effect of NEFL overexpression on the malignant phenotypes of ESCC cells, and the results showed that NEFL knockdown significantly impaired the ESCC cell invasion and migration in vitro. Xenograft assay in nude mice indicated that NEFL silencing suppressed tumor growth in vivo. At the molecular level, NEFL knockdown significantly upregulated E-cadherin and downregulated N-cadherin expression, suggesting that NEFL overexpression might influence the epithelial-mesenchymal transition (EMT) process. Furthermore, we found that NEFL knockdown significantly reduced the mRNA and protein expression of epidermal growth factor receptor (EGFR) and the phosphorylation levels of protein kinase B (PKB; also known as AKT) and ribosomal protein S6 (S6). Ectopic expression of EGFR after NEFL knockdown significantly restored the phosphorylation levels of AKT and S6 as well as the invasion and migration of ESCC cells. These data indicate that NEFL overexpression might promote the EMT process of ESCC cells via the EGFR/AKT/S6 pathway, ultimately enhancing the invasion and migration of ESCC cells.

FKBP10 promotes proliferation of glioma cells via activating AKT-CREB-PCNA axis.

BACKGROUND: Although the availability of therapeutic options including temozolomide, radiotherapy and some target agents following neurosurgery, the prognosis of glioma patients remains poor. Thus, there is an urgent need to explore possible targets for clinical treatment of this disease. METHODS: Tissue microarrays and immunohistochemistry were used to detect FKBP10, Hsp47, p-AKT (Ser473), p-CREB (Ser133) and PCNA expression in glioma tissues and xenografts. CCK-8 tests, colony formation assays and xenograft model were performed to test proliferation ability of FKBP10 in glioma cells in vitro and in vivo. Quantitative reverse transcriptase-PCR, western-blotting, GST-pull down, co-immunoprecipitation and confocal-immunofluorescence staining assay were used to explore the molecular mechanism underlying the functions of overexpressed FKBP10 in glioma cells. RESULTS: FKBP10 was highly expressed in glioma tissues and its expression was positively correlates with grade, poor prognosis. FKBP10-knockdown suppressed glioma cell proliferation in vitro and subcutaneous/orthotopic xenograft tumor growth in vivo. Silencing of FKBP10 reduced p-AKT (Ser473), p-CREB (Ser133), PCNA mRNA and PCNA protein expression in glioma cells. FKBP10 interacting with Hsp47 enhanced the proliferation ability of glioma cells via AKT-CREB-PCNA cascade. In addition, correlation between these molecules were also found in xenograft tumor and glioma tissues. CONCLUSIONS: We showed for the first time that FKBP10 is overexpressed in glioma and involved in proliferation of glioma cells by interacting with Hsp47 and activating AKT-CREB-PCNA signaling pathways. Our findings suggest that inhibition of FKBP10 related signaling might offer a potential therapeutic option for glioma patients.

Remarkable inhibition effects of afatinib alone or combining with paclitaxel in esophageal squamous cell carcinoma.

BACKGROUND AND AIM: Chemotherapy drugs do not work well in esophageal squamous cell carcinoma (ESCC), and none of the targeted drugs have been applied in clinic. This study aims to identify effective targeted drugs and related biomarkers for the treatment of ESCC. METHODS: The effect of 40 Food and Drug Administration-approved small-molecule inhibitors was first tested in five ESCC cell lines. CCK8 assays and xenografts derived from ESCC cell lines were performed to evaluate the anti-ESCC effects of inhibitors or chemotherapeutic agents in vitro and in vivo, respectively. Immunohistochemistry was utilized to analyze the p-EGFR expression in tissues. Western blot combining with gray analysis was conducted to detect the expression of interest protein. Flow cytometry and immunofluorescence assay were used to analyze apoptosis, cell cycle, and mitotic changes after drug treatment. RESULTS: Afatinib showed remarkable effects on inhibiting ESCC cells with higher expression of p-EGFR. Results from combinatorial screening in ESCC cells expressing lower phosphorylation level of EGFR showed that paclitaxel and afatinib presented a significant synergistic inhibitory effect (P < 0.001). Molecular analysis revealed that paclitaxel sensitized afatinib by activating EGFR, and afatinib in combination with paclitaxel effectively blocked MAPK pathway and induced G2/M cell arrest and apoptosis that is an indicator of mitotic catastrophe. CONCLUSIONS: Our data demonstrate that afatinib is an effective drug for patients with ESCC expressing higher phosphorylation level of EGFR. And for patients with lower p-EGFR in tumors, paclitaxel in combination with afatinib might be a promising therapeutic strategy in ESCC.

Elevated serum eotaxin and IP-10 levels as potential biomarkers for the detection of esophageal squamous cell carcinoma.

BACKGROUND AND AIMS: Esophageal squamous cell cancer (ESCC) is one of the leading malignant cancers with a high incidence and mortality. Exploring novel serum biomarkers will help improve the management and monitoring of ESCC. METHODS: In the present study, we first used a ProcartaPlex Array to screen for serum proteins that were increased in 40 ESCC patients compared with matched normal controls; we found that eight proteins (IL-2, IL-5, IP-10, IL-8, eotaxin, TNF-α, HGF, and MIP-1b) had higher serum levels in ESCC patients than in normal controls. We further verified the clinical relevance of the candidate biomarkers with a larger sample of sera. RESULTS: In the 174 tested ESCC patients and 189 normal controls, the serum levels of eotaxin and IP-10 were significantly higher in patients than in normal controls (p = 0.0038, 0.0031). In particular, these two proteins were also elevated in the sera of patients with early-stage (0-IIA) ESCC (p = 0.0041, 0.0412). When combining CEA and CYFRA21-1 (in use clinically) with eotaxin or IP-10, the effectiveness of detecting ESCC was superior to that of CEA and/or CYFRA21-1 alone. Moreover, the serum level of eotaxin dropped significantly after surgical resection of primary tumors compared with that in preoperative ESCC samples (p < 0.001). CONCLUSIONS: The data suggest that serum eotaxin and IP-10 might be potential biomarkers for the detection of ESCC.

Research advances of secretory proteins in malignant tumors.

Secretory proteins in tumor tissues are important components of the tumor microenvironment. Secretory proteins act on tumor cells or stromal cells or mediate interactions between tumor cells and stromal cells, thereby affecting tumor progression and clinical treatment efficacy. In this paper, recent research advances in secretory proteins in malignant tumors are reviewed.

PTP1B up-regulates EGFR expression by dephosphorylating MYH9 at Y1408 to promote cell migration and invasion in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common cancers worldwide. Protein tyrosine phosphatase 1B (PTP1B) is a member of protein tyrosine phosphatases (PTPs) family. In our previous work, PTP1B was found to be overexpressed in ESCC tissues and made contributions to the the cell migration and invasion as well as lung metastasis of ESCC. In this study, we explored the underlying molecular mechanisms. PTP1B enhanced cell migration and invasion by promoting epidermal growth factor receptor (EGFR) expression in ESCC, which was relied on phosphatase activity of PTP1B. Using GST-pulldown combined with LC/MS/MS, we found that nonmuscle myosin IIA (MYH9) was a novel substrate of PTP1B in ESCC cells. PTP1B dephosphorylated MYH9 at Y1408, by which PTP1B up-regulated EGFR expression and enhanced cell migration and invasion in ESCC. In conclusion, our study first reported that PTP1B was the positive regulator of EGFR by dephosphorylating MYH9 at Y1408 to promote cell migration and invasion, which revealed the regulatory mechanism of PTP1B-MYH9-EGFR axis in ESCC.

Identification of genomic alterations in oesophageal squamous cell cancer.

Oesophageal cancer is one of the most aggressive cancers and is the sixth leading cause of cancer death worldwide. Approximately 70% of global oesophageal cancer cases occur in China, with oesophageal squamous cell carcinoma (ESCC) being the histopathological form in the vast majority of cases (>90%). Currently, there are limited clinical approaches for the early diagnosis and treatment of ESCC, resulting in a 10% five-year survival rate for patients. However, the full repertoire of genomic events leading to the pathogenesis of ESCC remains unclear. Here we describe a comprehensive genomic analysis of 158 ESCC cases, as part of the International Cancer Genome Consortium research project. We conducted whole-genome sequencing in 17 ESCC cases and whole-exome sequencing in 71 cases, of which 53 cases, plus an additional 70 ESCC cases not used in the whole-genome and whole-exome sequencing, were subjected to array comparative genomic hybridization analysis. We identified eight significantly mutated genes, of which six are well known tumour-associated genes (TP53, RB1, CDKN2A, PIK3CA, NOTCH1, NFE2L2), and two have not previously been described in ESCC (ADAM29 and FAM135B). Notably, FAM135B is identified as a novel cancer-implicated gene as assayed for its ability to promote malignancy of ESCC cells. Additionally, MIR548K, a microRNA encoded in the amplified 11q13.3-13.4 region, is characterized as a novel oncogene, and functional assays demonstrate that MIR548K enhances malignant phenotypes of ESCC cells. Moreover, we have found that several important histone regulator genes (MLL2 (also called KMT2D), ASH1L, MLL3 (KMT2C), SETD1B, CREBBP and EP300) are frequently altered in ESCC. Pathway assessment reveals that somatic aberrations are mainly involved in the Wnt, cell cycle and Notch pathways. Genomic analyses suggest that ESCC and head and neck squamous cell carcinoma share some common pathogenic mechanisms, and ESCC development is associated with alcohol drinking. This study has explored novel biological markers and tumorigenic pathways that would greatly improve therapeutic strategies for ESCC.

Genomic and Epigenomic Aberrations in Esophageal Squamous Cell Carcinoma and Implications for Patients.

Esophageal squamous cell carcinoma (ESCC) is a common malignancy without effective therapy. The exomes of more than 600 ESCCs have been sequenced in the past 4 years, and numerous key aberrations have been identified. Recently, researchers reported both inter- and intratumor heterogeneity. Although these are interesting observations, their clinical implications are unclear due to the limited number of samples profiled. Epigenomic alterations, such as changes in DNA methylation, histone acetylation, and RNA editing, also have been observed in ESCCs. However, it is not clear what proportion of ESCC cells carry these epigenomic aberrations or how they contribute to tumor development. We review the genomic and epigenomic characteristics of ESCCs, with a focus on emerging themes. We discuss their clinical implications and future research directions.

Identification of distinct mutational patterns and new driver genes in oesophageal squamous cell carcinomas and adenocarcinomas.

OBJECTIVES: Oesophageal squamous cell carcinoma (OSCC) and adenocarcinoma (OAC) are distinct cancers in terms of a number of clinical and epidemiological characteristics, complicating the design of clinical trials and biomarker developments. We analysed 1048 oesophageal tumour-germline pairs from both subtypes, to characterise their genomic features, and biological and clinical significance. DESIGN: Previously exome-sequenced samples were re-analysed to identify significantly mutated genes (SMGs) and mutational signatures. The biological functions of novel SMGs were investigated using cell line and xenograft models. We further performed whole-genome bisulfite sequencing and chromatin immunoprecipitation (ChIP)-seq to characterise epigenetic alterations. RESULTS: OSCC and OAC displayed nearly mutually exclusive sets of driver genes, indicating that they follow independent developmental paths. The combined sample size allowed the statistical identification of a number of novel subtype-specific SMGs, mutational signatures and prognostic biomarkers. Particularly, we identified a novel mutational signature similar to Catalogue Of Somatic Mutations In Cancer (COSMIC)signature 16, which has prognostic value in OSCC. Two newly discovered SMGs, CUL3 and ZFP36L2, were validated as important tumour-suppressors specific to the OSCC subtype. We further identified their additional loss-of-function mechanisms. CUL3 was homozygously deleted specifically in OSCC and other squamous cell cancers (SCCs). Notably, ZFP36L2 is associated with super-enhancer in healthy oesophageal mucosa; DNA hypermethylation in its super-enhancer reduced active histone markers in squamous cancer cells, suggesting an epigenetic inactivation of a super-enhancer-associated SCC suppressor. CONCLUSIONS: These data comprehensively contrast differences between OSCC and OAC at both genomic and epigenomic levels, and reveal novel molecular features for further delineating the pathophysiological mechanisms and treatment strategies for these cancers.

Targeting super-enhancer-associated oncogenes in oesophageal squamous cell carcinoma.

OBJECTIVES: Oesophageal squamous cell carcinoma (OSCC) is an aggressive malignancy and the major histological subtype of oesophageal cancer. Although recent large-scale genomic analysis has improved the description of the genetic abnormalities of OSCC, few targetable genomic lesions have been identified, and no molecular therapy is available. This study aims to identify druggable candidates in this tumour. DESIGN: High-throughput small-molecule inhibitor screening was performed to identify potent anti-OSCC compounds. Whole-transcriptome sequencing (RNA-Seq) and chromatin immunoprecipitation sequencing (ChIP-Seq) were conducted to decipher the mechanisms of action of CDK7 inhibition in OSCC. A variety of in vitro and in vivo cellular assays were performed to determine the effects of candidate genes on OSCC malignant phenotypes. RESULTS: The unbiased high-throughput small-molecule inhibitor screening led us to discover a highly potent anti-OSCC compound, THZ1, a specific CDK7 inhibitor. RNA-Seq revealed that low-dose THZ1 treatment caused selective inhibition of a number of oncogenic transcripts. Notably, further characterisation of the genomic features of these THZ1-sensitive transcripts demonstrated that they were frequently associated with super-enhancer (SE). Moreover, SE analysis alone uncovered many OSCC lineage-specific master regulators. Finally, integrative analysis of both THZ1-sensitive and SE-associated transcripts identified a number of novel OSCC oncogenes, including PAK4, RUNX1, DNAJB1, SREBF2 and YAP1, with PAK4 being a potential druggable kinase. CONCLUSIONS: Our integrative approaches led to a catalogue of SE-associated master regulators and oncogenic transcripts, which may significantly promote both the understanding of OSCC biology and the development of more innovative therapies.

Cell-free ribosome display and selection of antibodies on arrayed antigens.

In vitro display technology is a powerful tool for discovery and optimisation of novel antibodies. With increasing demands on various binding molecules in proteomics studies, techniques for a large-scale generation of antibodies or antibody fragments are needed. Here, we describe a novel method for parallel generation of different antibody fragments (scFv) by integrating cell-free ribosome display with array technology. We have demonstrated the procedure by successfully isolating scFv antibodies specific to 16 different cancer biomarkers via a single process. Our results provide proof of principle for multiple production of various scFv antibodies simultaneously.

Novel role for non-homologous end joining in the formation of double minutes in methotrexate-resistant colon cancer cells.

BACKGROUND: Gene amplification is a frequent manifestation of genomic instability that plays a role in tumour progression and development of drug resistance. It is manifested cytogenetically as extrachromosomal double minutes (DMs) or intrachromosomal homogeneously staining regions (HSRs). To better understand the molecular mechanism by which HSRs and DMs are formed and how they relate to the development of methotrexate (MTX) resistance, we used two model systems of MTX-resistant HT-29 colon cancer cell lines harbouring amplified DHFR primarily in (i) HSRs and (ii) DMs. RESULTS: In DM-containing cells, we found increased expression of non-homologous end joining (NHEJ) proteins. Depletion or inhibition of DNA-PKcs, a key NHEJ protein, caused decreased DHFR amplification, disappearance of DMs, increased formation of micronuclei or nuclear buds, which correlated with the elimination of DHFR, and increased sensitivity to MTX. These findings indicate for the first time that NHEJ plays a specific role in DM formation, and that increased MTX sensitivity of DM-containing cells depleted of DNA-PKcs results from DHFR elimination. Conversely, in HSR-containing cells, we found no significant change in the expression of NHEJ proteins. Depletion of DNA-PKcs had no effect on DHFR amplification and resulted in only a modest increase in sensitivity to MTX. Interestingly, both DM-containing and HSR-containing cells exhibited decreased proliferation upon DNA-PKcs depletion. CONCLUSIONS: We demonstrate a novel specific role for NHEJ in the formation of DMs, but not HSRs, in MTX-resistant cells, and that NHEJ may be targeted for the treatment of MTX-resistant colon cancer.

Overexpression of DNAJB6 promotes colorectal cancer cell invasion through an IQGAP1/ERK-dependent signaling pathway.

DNAJB6 is a member of the heat shock protein 40 (Hsp40) family. We here investigated the clinical correlation and biological role of DNAJB6 overexpression in colorectal cancer (CRC). The expression of DNAJB6 protein was examined in 200 cases of colorectal adenocarcinomas by immunohistochemistry (IHC) technology. Gene transfection and RNA interference were performed to determine the effect of DNAJB6 expression on the invasion of CRC cells and to explore the underlying molecular mechanisms in vitro and in vivo. Overexpression of DNAJB6 was found in 39% (78/200) of the CRC tissues, especially in tumors at pT4 as compared with at pT1-3 (P = 0.02). A Kaplan-Meier survival analysis revealed a correlation between DNAJB6 expression and overall survival (OS) times (P = 0.003). Multivariate analysis confirmed that DNAJB6 overexpression was an independent prognostic factor for CRC (P = 0.002). RNA interference-mediated silencing of the DNAJB6 gene inhibited the invasion of CRC cells in vitro were accompanied by a significant reduction in the protein levels of IQ-domain GTPase-activating protein 1 (IQGAP1) and phosphorylated ERK (pERK). An in vivo assay showed that inhibition of DNAJB6 expression decreased the lung metastases of CRC cells. IHC analysis of serial sections showed that there was a positive correlation between DNAJB6 and IQGAP1 expression in primary CRC tissues (P = 0.013). The data suggest that DNAJB6 plays an important oncogenic role in CRC cell invasion by up-regulating IQGAP1 and activating the ERK signaling pathway and that DNAJB6 may be used as a prognostic marker for CRC.

Efficient expression, purification and characterization of native human cystatin C in Escherichia coli periplasm.

Human cystatin C (HCC), encoded by cystatin 3 gene, is a 13.3kDa endogenous cysteine proteinase inhibitor and an important biomarker of renal function. However, expressing recombinant cystatin C is difficult because of low yield and inclusion bodies in Escherichia coli (E. coli). In this study, we cloned HCC gene into pET-22b vector containing PelB leader signal sequence, which could direct the protein to the bacterial periplasm. Large amounts of soluble HCC could be efficiently expressed in the bacterial periplasm at 16°C with 0.1mM IPTG induction. The recombinant HCC was isolated in high purity by cation exchange chromatography and gel filtration chromatography. Furthermore, the HCC was characterized by circular dichroism (CD) and dynamic light scattering (DLS), and displayed biological activity against papain. Here, we provide a method to produce large amounts of soluble mature HCC in E. coli.

[IGHMBP2 overexpression promotes cell migration and invasion in esophageal squamous carcinoma].

Immunoglobulin mu binding protein 2 (IGHMBP2) is located in 11q13.2, which is frequently amplified in esophageal squamous cell carcinoma (ESCC). IGHMBP2 encodes a helicase involved in DNA replication and repair. IGHMBP2 protein also regulates gene transcription. The present study aims to explore the amplification of IGHMBP2 and its potential role in ESCC. A further analysis of our previously reported array-CGH data showed that IGHMBP2 was amplified in 28.9% of primary ESCC tumors. Fluorescence in situ hybridization (FISH) and Western blot showed that IGHMBP2 was amplified and overexpressed in KYSE30, KYSE180, KYSE510 and KYSE150 esophageal cancer cell lines. Transwell assays demonstrated that knockdown of IGHMBP2 in KYSE30 and KYSE150 inhibited cell invasion and migration, and increased the expression levels of E-cadherin. When rescue plasmids expressing IGHMBP2 were introduced, the abilities of cell invasion and migration were restored. These data suggest that IGHMBP2 overexpression may promote invasion and migration of ESCC cells through down-regulation of E-cadherin.

Genetic code-guided protein synthesis and folding in Escherichia coli.

Universal genetic codes are degenerated with 61 codons specifying 20 amino acids, thus creating synonymous codons for a single amino acid. Synonymous codons have been shown to affect protein properties in a given organism. To address this issue and explore how Escherichia coli selects its "codon-preferred" DNA template(s) for synthesis of proteins with required properties, we have designed synonymous codon libraries based on an antibody (scFv) sequence and carried out bacterial expression and screening for variants with altered properties. As a result, 342 codon variants have been identified, differing significantly in protein solubility and functionality while retaining the identical original amino acid sequence. The soluble expression level varied from completely insoluble aggregates to a soluble yield of ~2.5 mg/liter, whereas the antigen-binding activity changed from no binding at all to a binding affinity of > 10(-8) m. Not only does our work demonstrate the involvement of genetic codes in regulating protein synthesis and folding but it also provides a novel screening strategy for producing improved proteins without the need to substitute amino acids.

X-linked congenital hypertrichosis syndrome is associated with interchromosomal insertions mediated by a human-specific palindrome near SOX3.

X-linked congenital generalized hypertrichosis (CGH), an extremely rare condition characterized by universal overgrowth of terminal hair, was first mapped to chromosome Xq24-q27.1 in a Mexican family. However, the underlying genetic defect remains unknown. We ascertained a large Chinese family with an X-linked congenital hypertrichosis syndrome combining CGH, scoliosis, and spina bifida and mapped the disease locus to a 5.6 Mb critical region within the interval defined by the previously reported Mexican family. Through the combination of a high-resolution copy-number variation (CNV) scan and targeted genomic sequencing, we identified an interchromosomal insertion at Xq27.1 of a 125,577 bp intragenic fragment of COL23A1 on 5q35.3, with one X breakpoint within and the other very close to a human-specific short palindromic sequence located 82 kb downstream of SOX3. In the Mexican family, we found an interchromosomal insertion at the same Xq27.1 site of a 300,036 bp genomic fragment on 4q31.2, encompassing PRMT10 and TMEM184C and involving parts of ARHGAP10 and EDNRA. Notably, both of the two X breakpoints were within the short palindrome. The two palindrome-mediated insertions fully segregate with the CGH phenotype in each of the families, and the CNV gains of the respective autosomal genomic segments are not present in the public database and were not found in 1274 control individuals. Analysis of control individuals revealed deletions ranging from 173 bp to 9104 bp at the site of the insertions with no phenotypic consequence. Taken together, our results strongly support the pathogenicity of the identified insertions and establish X-linked congenital hypertrichosis syndrome as a genomic disorder.

Inhibition of atypical protein kinase Cι induces apoptosis through autophagic degradation of ß-catenin in esophageal cancer cells.

Atypical protein kinase Cι (PKCι) has been identified as an oncoprotein in esophageal squamous cell carcinomas. However, the mechanisms underlying the role of PKCι in this disease remain unclear. In the present work, we found that inhibition of PKCι expression by RNAi induced apoptosis via the down-regulation of ß-catenin in esophageal cancer cells. Furthermore, we found that PKCι regulated ß-catenin in an autophagy dependent way. Since down-regulation of ß-catenin induced by knockdown of PKCι could be rescued by autophagy inhibition; knockdown of PKCι activated autophagy and promoted the recruitment of ß-catenin into autophagosome. These results suggested that PKCι positively regulated ß-catenin through negatively regulated autophagy and depletion of PKCι promoted apoptosis via autophagic degradation of ß-catenin in esophageal cancer cells. These data provide new insights into PKCι signaling in human cancer.