Gene-dosage dependent overexpression at the 13q amplicon identifies DIS3 as candidate oncogene in colorectal cancer progression.

Colorectal cancer (CRC) development is in most cases marked by the accumulation of genomic alterations including gain of the entire q-arm of chromosome 13. This aberration occurs in 40%-60% of all CRC and is associated with progression from adenoma to carcinoma. To date, little is known about the effect of the 13q amplicon on the expression of the therein located genes and their functional relevance. We therefore aimed to identify candidate genes at the 13q amplicon that contribute to colorectal adenoma to carcinoma progression in a gene dosage-dependent manner. Integrative analysis of whole genome expression and DNA copy number signatures resulted in the identification of 36 genes on 13q of which significant overexpression in carcinomas compared with adenomas was linked to a copy number gain. Five genes showing high levels of overexpression in carcinomas versus adenomas were further tested by quantitative reverse transcription-PCR in two independent sample sets of colorectal tumors (n = 40 and n = 47). DIS3 and LRCH1 revealed significant overexpression in carcinomas compared with adenomas in a 13q gain dependent manner. Silencing of DIS3 affected important tumorigenic characteristics such as viability, migration, and invasion. In conclusion, significant overexpression of DIS3 and LRCH1 associated with adenoma to carcinoma progression is linked to the CRC specific gain of 13q. The functional relevance of this copy number aberration was corroborated for DIS3, thereby identifying this gene as novel candidate oncogene contributing to the 13q-driven adenoma to carcinoma progression.

Molecular genetic analysis of vesicular transport in Aspergillus niger reveals partial conservation of the molecular mechanism of exocytosis in fungi.

The filamentous fungus Aspergillus niger is an industrially exploited protein expression platform, well known for its capacity to secrete high levels of proteins. To study the process of protein secretion in A. niger, we established a GFP-v-SNARE reporter strain in which the trafficking and dynamics of secretory vesicles can be followed in vivo. The biological role of putative A. niger orthologues of seven secretion-specific genes, known to function in key aspects of the protein secretion machinery in Saccharomyces cerevisiae, was analysed by constructing respective gene deletion mutants in the GFP-v-SNARE reporter strain. Comparison of the deletion phenotype of conserved proteins functioning in the secretory pathway revealed common features but also interesting differences between S. cerevisiae and A. niger. Deletion of the S. cerevisiae Sec2p orthologue in A. niger (SecB), encoding a guanine exchange factor for the GTPase Sec4p (SrgA in A. niger), did not have an obvious phenotype, while SEC2 deletion in S. cerevisiae is lethal. Similarly, deletion of the A. niger orthologue of the S. cerevisiae exocyst subunit Sec3p (SecC) did not result in a lethal phenotype as in S. cerevisiae, although severe growth reduction of A. niger was observed. Deletion of secA, secH and ssoA (encoding SecA, SecH and SsoA the A. niger orthologues of S. cerevisiae Sec1p, Sec8p and Sso1/2p, respectively) showed that these genes are essential for A. niger, similar to the situation in S. cerevisiae. These data demonstrate that the orchestration of exocyst-mediated vesicle transport is only partially conserved in S. cerevisiae and A. niger.

Overexpression of the miR-17-92 cluster in colorectal adenoma organoids causes a carcinoma-like gene expression signature.

Gain of chromosome arm 13q is one of the most prevalent DNA copy number alterations associated with colorectal adenoma-to-carcinoma progression. The oncogenic miR-17-92 cluster, located at 13q, was found to be overexpressed in colorectal cancer and in adenomas harboring 13q gain. However, to what extent overexpression of this group of microRNAs actually drives progression to cancer remains to be resolved. Therefore, we aimed to clarify the role of miR-17-92 cluster in the progression from colorectal adenoma to carcinoma. The miR-17-92 cluster was overexpressed in human colorectal adenoma organoids without 13q gain and downstream effects on mRNA expression were investigated, along with functional consequences in vitro and in vivo. Comparison of mRNA sequencing results of organoids overexpressing miR-17-92 and cultures transduced with control vector revealed a miR-17-92 expression signature. This signature appeared to be enriched in an independent series of colorectal cancers and adenomas with 13q gain, confirming that miR-17-92 expression is associated with malignant progression. However, tumor-associated characteristics, such as increased proliferation rate, were not observed in miR-17-92 overexpressing adenoma organoids in vitro. In addition, subcutaneous injection of these organoids in immunodeficient mice was insufficient to cause tumor outgrowth. In conclusion, this study showed that miR-17-92 expression contributes to 13q gain-associated adenoma-to-carcinoma progression, however, this is insufficient to cause malignancy.

Oncogenic Role of miR-15a-3p in 13q Amplicon-Driven Colorectal Adenoma-to-Carcinoma Progression.

Progression from colorectal adenoma to carcinoma is strongly associated with an accumulation of genomic alterations, including gain of chromosome 13. This gain affects the whole q arm and is present in 40%-60% of all colorectal cancers (CRCs). Several genes located at this amplicon are known to be overexpressed in carcinomas due to copy number dosage. A subset of these genes, including the mir-17~92 cluster, are functionally involved in CRC development. The present study set out to explore whether apart from mir-17~92, other miRNAs located at the 13q amplicon show a copy number dependent dosage effect that may contribute to 13q-driven colorectal adenoma-to-carcinoma progression. Integration of publically available miRNA expression, target mRNA expression and DNA copy number data from 125 CRCs yielded three miRNAs, miR-15a, -17, and -20a, of which high expression levels were significantly correlated with a 13q gain and which influenced target mRNA expression. These results could be confirmed by qRT-PCR in a series of 100 colon adenomas and carcinomas.Functional analysis of both mature miRNAs encoded by mir-15a, i.e. miR-15a-5p and miR-15a-3p, showed that silencing of miR-15a-3p significantly inhibited viability of CRC cells. Integration of miR-15a expression levels with mRNA expression data of predicted target genes identified mitochondrial uncoupling protein 2 (UCP2) and COP9 signalosome subunit 2 (COPS2) as candidates with significantly decreased expression in CRCs with 13q gain. Upon silencing of miR-15a-3p, mRNA expression of both genes increased in CRC cells, supporting miR-15a-3p mediated regulation of UPC2 and COPS2 expression. In conclusion, significant overexpression of miR-15a-3p due to gain of 13q is functionally relevant in CRC, with UCP2 and COPS2 as candidate target genes. Taken together our findings suggest that miR-15a-3p may contribute to adenoma-to-carcinoma progression.