Rac1 GTPase and the Rac1 exchange factor Tiam1 associate with Wnt-responsive promoters to enhance beta-catenin/TCF-dependent transcription in colorectal cancer cells

<p>Abstract</p> <p>Background</p> <p>β-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with β-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the β-catenin/TCF transcription factor complex.</p> <p>Results</p> <p>We employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the β-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and β-catenin. Furthermore, endogenous Tiam1 associated with endogenous β-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner.</p> <p>Conclusion</p> <p>Taken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active β-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.</p

Tumor BRCA Testing in High Grade Serous Carcinoma: Mutation Rates and Optimal Tissue Requirements

Background: Approximately 25% of women diagnosed with tubo-ovarian high-grade serous carcinoma have germline deleterious mutations in BRCA1 or BRCA2, characteristic of hereditary breast and ovarian cancer syndrome, while somatic mutations have been detected in 3–7%. We set out to determine the BRCA mutation rates and optimal tissue requirements for tumor BRCA testing in patients diagnosed with tubo-ovarian high-grade serous carcinoma. Methods: Sequencing was performed using a multiplexed polymerase chain reaction-based approach on 291 tissue samples, with a minimum sequencing depth of 500X and an allele frequency of >5%. Results: There were 253 surgical samples (87%), 35 biopsies (12%) and 3 cytology cell blocks (1%). The initial failure rate was 9% (25/291), including 9 cases (3%) with insufficient tumor, and 16 (6%) with non-amplifiable DNA. Sequencing was successful in 78% (228/291) and deemed indeterminate due to failed exons or variants below the limit of detection in 13% (38/291). Repeat testing was successful in 67% (28/42) of retested samples, with an overall success rate of 86% (251/291). Clinically significant (pathogenic, likely pathogenic) variants were identified in 17% (48/276) of complete and indeterminate cases. Successful sequencing was dependent on sample type, tumor cellularity and size (p ≤ 0.001) but not on neoadjuvant chemotherapy or age of blocks (p > 0.05). Conclusions: Our study shows a 17% tumor BRCA mutation rate, with an overall success rate of 86%. Biopsy and cytology samples and post-chemotherapy specimens can be used for tumor BRCA testing, and optimal tumors measure ≥5 mm in size with at least 20% cellularity

Rare mutations predisposing to familial adenomatous polyposis in Greek FAP patients

BACKGROUND: Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions / deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. METHODS: In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation – dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription – Polymerase Chain Reaction). RESULTS: A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. CONCLUSION: Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations

Molecular Mechanisms of the Cooperation between Rac1/1b GTPases and the Canonical Wnt Signaling Pathway in Colorectal Cancer

Aberrant activation of the canonical Wnt signaling pathway accounts for the vast majority of colorectal cancers. The Rac1 GTPase is overexpressed in colon cancer, and its splice variant, Rac1b, is preferentially expressed in colon tumours. Rac1 and Rac1b have both been previously shown to crosstalk with the canonical Wnt signaling pathway in colon cancer; however, the specific means by which this crosstalk occurs were unclear. This study examines the molecular mechanisms of Rac1/1b in the cooperation with canonical Wnt signaling in colon cancer. In a colon cancer cell line with dysregulated Wnt signaling, the constitutively active Rac1 mutant, V12Rac1, was observed to transcriptionally upregulate the expression of a gene set associated with cellular migration. Further, V12Rac1-mediated promotion of cell migration was dependent on its nuclear localization. Previous work in our lab has shown a Rac1-specific activator, Tiam1, is present in the nucleus at the promoter of Wnt target genes upon Wnt3a stimulation; and that exogenous introduction of Tiam1 increased the expression of a Wnt-responsive reporter (TopFlash). Given the importance of nuclear localization of Rac1 in the promotion of tumourigenic processes, we demonstrated that knockdown of endogenous Tiam1 reduced TopFlash expression, proving reverse specificity and strengthening the evidence of a nuclear role for Rac1. Since some functional differences exist between Rac1 and Rac1b, we also examined Rac1b for transcriptional targets following induction, and identified the RhoA effector, ROCK2, which has been previously associated with cell migration. ROCK2 demonstrated a positive correlation with Rac1b transcript expression in primary colon tumours as compared to matched normal tissue specimens. Interestingly, the observed induction in ROCK2 transcript did not translate into a detectable change in protein expression or kinase activity. Like Rac1, Rac1b also promotes cellular motility, which is dependent on nuclear localization. Cell migration can be negatively regulated by E-cadherin. Following Rac1b knockdown in HT29 cells, we show that Rac1b might contribute to motility through upregulation of the E-cadherin-repressor, Slug. Taken together, we provide greater insight into the mechanistic roles of Rac1 and Rac1b in transcriptionally regulating target genes to promote cellular processes, such as cell migration, in colon cancer with dysregulated canonical Wnt signaling.Ph

The GoodHope Ehlers Danlos Syndrome Clinic: development and implementation of the first interdisciplinary program for multi-system issues in connective tissue disorders at the Toronto General Hospital

Abstract Ehlers-Danlos Syndrome (EDS) are a heterogeneous group of genetic connective tissue disorders, and typically manifests as weak joints that subluxate/dislocate, stretchy and/or fragile skin, organ/systems dysfunction, and significant widespread pain. Historically, this syndrome has been poorly understood and often overlooked. As a result, people living with EDS had difficulty obtaining an accurate diagnosis and appropriate treatment, leading to untold personal suffering as well as ineffective health care utilization. The GoodHope EDS clinic addresses systemic gaps in the diagnosis and treatment of EDS. This paper describes a leap forward—from lack of awareness, diagnosis, and treatment—to expert care that is tailored to meet the specific needs of patients with EDS. The GoodHope EDS clinic consists of experts from various medical specialties who work together to provide comprehensive care that addresses the multi-systemic nature of the syndrome. In addition, EDS-specific self-management programs have been developed that draw on exercise science, rehabilitation, and health psychology to improve physical and psychosocial wellbeing and overall quality of life. Embedded into the program are research initiatives to shed light on the clinical presentation, underlying mechanisms of pathophysiology, and syndrome management. We also lead regular educational activities for community health care providers to increase awareness and competence in the interprofessional management of EDS beyond our doors and throughout the province and country

Combined hereditary and somatic mutations of replication error repair genes result in rapid onset of ultra-hypermutated cancers

DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase ɛ or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (∼600 mutations/cell division), reaching but not exceeding ∼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.status: publishe