Deletion of the WNT target and cancer stem cell marker CD44 in Apc(Min/+) mice attenuates intestinal tumorigenesis

Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or beta-catenin plays a critical role in the initiation of colorectal cancer. These mutations cause constitutively active beta-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to colorectal cancer precursor lesions, called dysplastic aberrant crypt foci. CD44 is a prominent WNT signaling target in the intestine and is selectively expressed on the renewing epithelial cells lining the crypts. The expression of CD44 is dramatically increased in aberrant crypt foci in both humans and tumor-susceptible Apc(Min/+) mice, suggesting a role for CD44 in intestinal tumorigenesis. To study this role, we crossed C57BL/6J-Cd44(-/-) mice with C57BL/6J-Apc(Min/+) mice. Compared with C57BL/6J-Cd44(+/+)/Apc(Min/+) mice, C57BL/6J-Cd44(-/-)/Apc(Min/+) mice showed an almost 50% reduction in the number of intestinal adenomas. This reduction was primarily caused by a decrease in the formation of aberrant crypts, implying the involvement of CD44 in tumor initiation. The absence of CD44 in the normal (nonneoplastic) crypts of Cd44(-/-)/Apc(Min/+) mice did not alter the proliferative capacity and size of the intestinal stem cell and transit-amplifying compartments. However, compared with Cd44(+/+)/Apc(Min/+) mice, Cd44(-/-)/Apc(Min/+) showed an increase in the number of apoptotic epithelial cells at the base of the crypt which correlated with an increased expression of the proapoptotic genes Bok and Dr6. Our results show an important role for CD44 in intestinal tumorigenesis and suggest that CD44 does not affect proliferation but is involved in the control of the balance between survival and apoptosis in the intestinal cryp

Differences in cartilage-forming capacity of expanded human chondrocytes from ear and nose and their gene expression profiles

The aim of this study was to evaluate the potential of culture-expanded human auricular and nasoseptal chondrocytes as cell source for regeneration of stable cartilage and to analyze the differences in gene expression profile of expanded chondrocytes from these specific locations. Auricular chondrocytes in monolayer proliferated less and more slowly (two passages took 26.7 ± 2.1 days and were reached in 4.37 ± 0.30 population doublings) than nasoseptal chondrocytes (19.3 ± 2.5 days; 5.45 ± 0.20 population doublings). However, auricular chondrocytes produced larger pellets with more cartilage-like matrix than nasoseptal chondrocytes (2.2 ± 0.71 vs. 1.7 ± 0.13 mm in diameter after 35 days of culture). Although the matrix formed by auricular and nasoseptal chondrocytes contained collagen X, it did not mineralize in an in vitro model or after in vivo subcutaneous implantation. A DNA microarray study on expanded auricular and nasoseptal chondrocytes from the same donors revealed 1,090 differentially expressed genes. No difference was observed in the expression of known markers of chondrogenic capacity (e.g., collagen II, FGFR3, BMP2, and ALK1). The most striking differences were that the auricular chondrocytes had a higher expression of anabolic growth factors BMP5 and IGF1, while matrix-degrading enzymes MMP13 and ADAMTS5 were higher expressed in nasoseptal chondrocytes. This might offer a possible explanation for the observed higher matrix production by auricular chondrocytes. Moreover, chondrocytes isolated from auricular or nasoseptal cartilage had specific gene expression profiles even after expansion. These differently expressed genes were not restricted to known characterization of donor site subtype (e.g., elastic), but were also related to developmental processe

Systematic discovery of gene fusions in pediatric cancer by integrating RNA-seq and WGS

Abstract Background Gene fusions are important cancer drivers in pediatric cancer and their accurate detection is essential for diagnosis and treatment. Clinical decision-making requires high confidence and precision of detection. Recent developments show RNA sequencing (RNA-seq) is promising for genome-wide detection of fusion products but hindered by many false positives that require extensive manual curation and impede discovery of pathogenic fusions. Methods We developed Fusion-sq to overcome existing disadvantages of detecting gene fusions. Fusion-sq integrates and “fuses” evidence from RNA-seq and whole genome sequencing (WGS) using intron–exon gene structure to identify tumor-specific protein coding gene fusions. Fusion-sq was then applied to the data generated from a pediatric pan-cancer cohort of 128 patients by WGS and RNA sequencing. Results In a pediatric pan-cancer cohort of 128 patients, we identified 155 high confidence tumor-specific gene fusions and their underlying structural variants (SVs). This includes all clinically relevant fusions known to be present in this cohort (30 patients). Fusion-sq distinguishes healthy-occurring from tumor-specific fusions and resolves fusions in amplified regions and copy number unstable genomes. A high gene fusion burden is associated with copy number instability. We identified 27 potentially pathogenic fusions involving oncogenes or tumor-suppressor genes characterized by underlying SVs, in some cases leading to expression changes indicative of activating or disruptive effects. Conclusions Our results indicate how clinically relevant and potentially pathogenic gene fusions can be identified and their functional effects investigated by combining WGS and RNA-seq. Integrating RNA fusion predictions with underlying SVs advances fusion detection beyond extensive manual filtering. Taken together, we developed a method for identifying candidate gene fusions that is suitable for precision oncology applications. Our method provides multi-omics evidence for assessing the pathogenicity of tumor-specific gene fusions for future clinical decision making

Molecular Characterization Reveals Subclasses of 1q Gain in Intermediate Risk Wilms Tumors

Chromosomal alterations have recurrently been identified in Wilms tumors (WTs) and some are associated with poor prognosis. Gain of 1q (1q+) is of special interest given its high prevalence and is currently actively studied for its prognostic value. However, the underlying mutational mechanisms and functional effects remain unknown. In a national unbiased cohort of 30 primary WTs, we integrated somatic SNVs, CNs and SVs with expression data and distinguished four clusters characterized by affected biological processes: muscle differentiation, immune system, kidney development and proliferation. Combined genome-wide CN and SV profiles showed that tumors profoundly differ in both their types of 1q+ and genomic stability and can be grouped into WTs with co-occurring 1p−/1q+, multiple chromosomal gains or CN neutral tumors. We identified 1q+ in eight tumors that differ in mutational mechanisms, subsequent rearrangements and genomic contexts. Moreover, 1q+ tumors were present in all four expression clusters reflecting activation of various biological processes, and individual tumors overexpress different genes on 1q. In conclusion, by integrating CNs, SVs and gene expression, we identified subgroups of 1q+ tumors reflecting differences in the functional effect of 1q gain, indicating that expression data is likely needed for further risk stratification of 1q+ WTs

Germline deletions in the tumour suppressor gene FOCAD are associated with polyposis and colorectal cancer development

Heritable genetic variants can significantly affect the lifetime risk of developing cancer, including polyposis and colorectal cancer (CRC). Variants in genes currently known to be associated with a high risk for polyposis or CRC, however, explain only a limited number of hereditary cases. The identification of additional genetic causes is, therefore, crucial to improve CRC prevention, detection and treatment. We have performed genome-wide and targeted DNA copy number profiling and resequencing in early-onset and familial polyposis/CRC patients, and show that deletions affecting the open reading frame of the tumour suppressor gene FOCAD are recurrent and significantly enriched in CRC patients compared with unaffected controls. All patients carrying FOCAD deletions exhibited a personal or family history of polyposis. RNA in situ hybridization revealed FOCAD expression in epithelial cells in the colonic crypt, the site of tumour initiation, as well as in colonic tumours and organoids. Our data suggest that monoallelic germline deletions in the tumour suppressor gene FOCAD underlie moderate genetic predisposition to the development of polyposis and CRC

A germline homozygous mutation in the base-excision repair gene NTHL1 causes adenomatous polyposis and colorectal cancer

The genetic cause underlying the development of multiple colonic adenomas, the premalignant precursors of colorectal cancer (CRC), frequently remains unresolved in patients with adenomatous polyposis. Here we applied whole-exome sequencing to 51 individuals with multiple colonic adenomas from 48 families. In seven affected individuals from three unrelated families, we identified a homozygous germline nonsense mutation in the base-excision repair (BER) gene NTHL1. This mutation was exclusively found in a heterozygous state in controls (minor allele frequency of 0.0036; n = 2,329). All three families showed recessive inheritance of the adenomatous polyposis phenotype and progression to CRC in at least one member. All three affected women developed an endometrial malignancy or premalignancy. Genetic analysis of three carcinomas and five adenomas from different affected individuals showed a non-hypermutated profile enriched for cytosine-to-thymine transitions. We conclude that a homozygous loss-of-function germline mutation in the NTHL1 gene predisposes to a new subtype of BER-associated adenomatous polyposis and CRC